Compounds that modulate fatty acid receptor activity and pet food products containing the same

ABSTRACT

A flavor composition comprising at least one compound that modulates, increases and/or enhances the activity of a GPR120 fatty acid receptor that can be used to enhance the fatty acid taste and/or palatability of pet food products is described herein. Also disclosed herein are methods for identifying said compounds.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a U.S. National Stage Patent Application under 35U.S.C. § 371 of International Application No. PCT/US2015/065106, filedon Dec. 10, 2015, which claims priority to U.S. Provisional ApplicationNo. 62/090,138 filed Dec. 10, 2014, the contents of each of which arehereby incorporated by reference in their entireties, and to whichpriority is claimed.

FIELD

The presently disclosed subject matter relates to compounds and flavorcompositions that include at least one compound that interacts with afatty acid receptor. The flavor compositions can be used to enhance ormodify the palatability, taste and/or flavor of pet food products. Theflavor compositions can include combinations of compounds, and can beadded to pet food products in various delivery system formats.

BACKGROUND

Taste profiles for edible compositions include basic tastes such assweet, salt, bitter, sour, umami and kokumi. Taste profiles have alsobeen described as including free fatty acid tastes. Chemical compoundsthat elicit these tastes are often referred to as tastants. It ishypothesized that tastants are sensed by taste receptors in the mouthand throat which transmit signals to the brain where the tastants andresulting taste profiles are registered. Taste receptors include theGPR120 (also known as GPR129, O3FAR1, PGR4, FFAR4) taste receptor, whichis predicted to be a G-protein coupled cell surface receptor, containingseven transmembrane domains involved in the detection of specific fattyacids, and the G-protein associated intracellular portion involved insignal transduction. GPR120 is thought to bind medium to long-chainfatty acids, such as oleic acid and linoleic acid, in their free form.It has been predicted that two isoforms (splice variants) of the GPR120receptor exists in humans, GPR120L and GPR120S, on colonic endocrinecells. It has been suggested that the long isoform does not signalfunctionally in the perception of taste.

GPR120 is expressed in various mammalian tissues and is a known receptorfor unsaturated long chain fatty acids. GPR120 has been known to beinvolved in the stimulation of cholecystokinin (CCK) secretion fromSTC-1, an intestinal secretory cell line, and it has been reported thatGPR120 has stimulatory effects on the secretion of glucogon like peptide(GLP 1). Although the potential involvement of GPR120 in stressregulation and insulin sensitization has been explored, as well as itspotential anti-inflammatory and anti-obesity effects (see, e.g., Cartoniet al., J Neurosci. 2010 Jun. 23; 30(25):8376-82; Céline et al., PLoSOne. 2011; 6(8):e24014; International Publication No. WO/2007/134613,published Nov. 29, 2007; European Publication No. EP1932920A1, publishedJun. 18, 2008; and European Publication No. EP1688138A1, published Aug.9, 2006), GPR120 has not been suggested to be involved in modulating thepalatability of pet food, and cat and dog food in particular.

Pet food manufacturers have a long-standing desire to provide pet foodproducts that have high nutritional value. In addition, and withparticular regard to cat and dog foods, pet food manufacturers desire ahigh degree of palatability so that pets can receive the fullnutritional benefit from their food. Domestic animals, especially cats,are notoriously fickle in their food preferences, and often refuse toeat a pet food product that it has accepted over some time or refuse toeat any more than a minimal amount of a pet food product. As a result,pet owners frequently change types and brands of pet food in order tomaintain their pets in a healthy and contented condition.

While there have been recent advances in taste and flavor technologies,there remains a need for compounds that can enhance or modify thepalatability of pet food products by enhancing or modifying the taste,texture and/or flavor profiles of the pet food product. The enhancementor modification can be to increase the intensity of a desirableattribute, to replace a desirable attribute that is not present orsomehow lost in the pet food product, or to decrease the intensity of anundesirable attribute. In particular, it is desirable to increase theintensity of a tastant in a pet food product. Therefore, there remains aneed in the art for compositions to enhance the palatability and tasteof pet food products, which is separate from any attempts to controlobesity or control fatty acid uptake.

SUMMARY OF THE INVENTION

The presently disclosed subject matter is directed to flavorcompositions and methods for making and modifying such compositionsacross a variety of pet food products. Specifically, the presentdisclosure is directed to compositions comprising one or more compoundsthat enhance, increase and/or modulate the activity of the fatty acidreceptor, GPR120.

In certain embodiments of the present disclosure, the flavor compositioncomprises one or more compound selected from the group consisting of

(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoic acid (also known asArachidonic Acid);

(5Z,8Z)-icosa-5,8-dienoic acid;

4-[4-(heptyloxy)phenyl]-4-oxobutanoic acid;

(11Z)-octadec-11-enoic acid (also known as cis-Vaccenic acid);

(9E)-hexadec-9-enoic acid (also known as Palmitelaidic acid);

tridec-12-enoic acid (also known as 12-Tridecenoic acid);

S-Farnesyl Thioacetic Acid;

(10Z)-pentadec-10-enoic acid (also known as (10Z)-10-Pentadecenoicacid);

10(E),12(Z)-Conjugated Linoleic Acid (also known as(10Z,12Z)-10,12-Octadecadienoic acid);

(10Z,13Z)-nonadeca-10,13-dienoic acid;

(9Z,11E)-octadeca-9,11-dienoic acid;

cis-7-Hexadecenoic Acid;

dodecanoic acid (also known as Lauric acid);

(9Z)-tetradec-9-enoic acid (also known as Myristoleic acid);

(11Z,14Z,17Z)-icosa-11,14,17-trienoic acid (also known asDihomo-α-linolenic acid (20:3(n-3)));

(6Z,9Z,12Z)-octadeca-6,9,12-trienoic acid (also known as γ-Linolenicacid);

(11Z,14Z)-icosa-11,14-dienoic acid (also known as Dihomolinoleic acid(20:2(n-6)));

(9Z)-hexadec-9-enoic acid (also known as palmitoleate, (Z)-Palmitoleicacid);

12-methoxydodecanoic acid;

(8Z,11Z,14Z)-icosa-8,11,14-trienoic acid;

(9Z,12Z)-octadeca-9,12-dienoic acid (also known as Linoleic acid);

(10Z)-heptadec-10-enoic acid;

Pinolenic Acid;

(9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid (also known as α-Linolenicacid);

tridecanoic acid (also known as Tridecylic acid);

tetradecanoic acid (also known as Myristic acid);

(9Z)-octadec-9-enoic acid (also known as Oleic acid);

GW 9508 (also known as4-[[(3-Phenoxyphenyl)methyl]amino]benzenepropanoic acid);

(5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenoic acid (also known asEicosapentanoic acid);

3-(4-((4-fluoro-4′-methylbiphenyl-2-yl)methoxy)phenyl)propanoic acid(also known as TUG 891 and4-[(4-Fluoro-4′-methyl[1,1′-biphenyl]-2-yl)methoxy]-benzenepropanoicacid);

(10E)-pentadec-10-enoic acid;

(9E)-tetradec-9-enoic acid (also known as Myristoleate);

(6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraenoic acid;

(5Z,8Z,11Z)-icosa-5,8,11-trienoic acid (also known as Mead acid); and

Dodecyl dihydrogen phosphate.

In certain embodiments of the present disclosure, the flavor compositioncomprises a compound of Formula I comprising the structure:

wherein R is a substituted or unsubstituted hydrocarbon chain.

In certain embodiments of the present disclosure, the flavor compositioncomprises a compound of Formula II comprising the structure:

wherein X, X₁, Y, W, Z₁, Z₂, n₁, n₂, and n₃ are as described herein.

In certain embodiments of the present disclosure, the flavor compositioncomprises a compound of Formula III comprising the structure:

wherein X, X₁, Y, W, R, n₁, n₂, and n₃ are as described herein.

In certain embodiments of the present disclosure, the flavor compositioncomprises a compound having the structure, R—X, wherein X is a carboxylgroup or a bioisosteric replacement for a caboxylate, and R is asubstituted or unsubstituted hydrocarbon chain.

The present disclosure also provides for salts and stereoisomers of thecompounds described herein.

In certain embodiments, the flavor composition further comprises atleast one, two, three, four, five or more first amino acids and/or atleast one, two, three, four, five or more second amino acids.Non-limiting examples of a first amino acid include tryptophan,phenylalanine, histidine, glycine, cysteine, alanine, tyrosine, serine,methionine, asparagine and leucine. Non-limiting examples of a secondamino acid include threonine, isoleucine, proline, glutamic acid,aspartic acid, hydroxyl proline, arginine, cystine, glutamine, lysine,valine, orthinine, monosodium glutamate (MSG) and taurine.

In certain embodiments, the flavor composition can further comprise atleast one standard nucleotide. Non-limiting examples of a standardnucleotide includes guanosine monophosphate (GMP), adenosinemonophosphate (AMP), cytidine monophosphate (CMP), inosine monophosphate(IMP), uridine monophosphate (UMP), thymidine monophosphate (TMP) andxanthosine monophosphate (XMP). In certain embodiments, the flavorcomposition can further comprise at least one nucleotide derivative, asdescribed herein.

In certain embodiments, the flavor composition can further comprise atleast one transmembrane compound, as described herein.

In certain embodiments, the present disclosure provides pet foodproducts including a flavor composition, comprising a compound, whereinthe flavor composition is present in an amount effective to increase afatty acid taste of the food products, as determined by a panel of tastetesters.

In certain embodiments, the present disclosure provides pet foodproducts including a flavor composition, comprising a compound, whereinthe flavor composition is present at a concentration of about 0.001weight % to about 10 weight % (% w/w), or about 0.01% to about 1% w/w ofthe pet food product. In certain embodiments, the pet food product is afeline pet food product.

In certain embodiments, the present disclosure provides pet foodproducts including a flavor composition, comprising a compound. Incertain embodiments, the flavor composition is present at aconcentration of about 0.01 ppm to about 1,000 ppm of the pet foodproduct. Alternatively or additionally, the compound can be present at aconcentration of about 10 pM to about 1 M in the pet food product.

The present disclosure further provides methods for increasing thepalatability of a pet food product. In certain embodiments, the methodcomprises admixing the pet food product with a flavor composition. Incertain embodiments, the flavor composition is present at aconcentration of about 0.001 weight % to about 10 weight % of theadmixture.

In certain embodiments of the present disclosure, a method forincreasing the palatability of a pet food product comprises admixing thepet food product with a flavor composition. In certain embodiments, theflavor composition is present at a concentration of about 0.01 ppm toabout 1,000 ppm of the admixture. Alternatively or additionally, the atleast one compound is present at a concentration of about 10 pM to about1 M in the admixture.

In certain embodiments of the present disclosure, a flavor compositionis admixed with a pet food product in an amount effective to increasethe palatability of the pet food product.

The presently disclosed subject matter also provides for methods ofmodulating the activity of a fatty acid receptor, comprising contactinga composition with a fatty acid receptor, for example, a feline fattyacid receptor comprising an amino acid sequence of SEQ ID NO: 1, whereinthe composition interacts with one or more amino acids in an interactingsite of the fatty acid receptor selected from the group consisting ofPHE88, VAL95, VAL98 and ARG99 on Helix 2; PHE115, MET118, SER119,GLY122, SER123 on Helix 3; TRP207, PHE211, VAL212, ASN215 on Helix 5;TRP277, ILE280, ILE281, ILE284 on Helix 6; and TRP299, PHE303, PHE304,VAL307, THR310, PHE311 on Helix 7; and combinations thereof.

The presently disclosed subject matter also provides for methods foridentifying a composition that modulates the activity of a fatty acidreceptor comprising contacting a test agent with a fatty acid receptorand detecting an interaction between the test agent and one or moreamino acids in an interacting site of the fatty acid receptor asdescribed herein.

The foregoing has outlined rather broadly the features and technicaladvantages of the present application in order that the detaileddescription that follows may be better understood. Additional featuresand advantages of the application will be described hereinafter whichform the subject of the claims of the application. It should beappreciated by those skilled in the art that the conception and specificembodiment disclosed may be readily utilized as a basis for modifying ordesigning other structures for carrying out the same purposes of thepresent application. It should also be realized by those skilled in theart that such equivalent constructions do not depart from the spirit andscope of the application as set forth in the appended claims. The novelfeatures which are believed to be characteristic of the application,both as to its organization and method of operation, together withfurther objects and advantages will be better understood from thefollowing description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1AH show in vitro fGPR120 receptor activity levels for threedifferent doses of various compounds, as described by Example 1.

FIGS. 2A-2AH show dose response curves for the in vitro activation offGPR120 for various compounds, as described by Example 1.

FIGS. 3A-3G show the chemical structures of the compounds described byExample 1, and FIGS. 1A-1AH and 2A-2AH, that increase fGPR120 activity.The number of carbon atoms in each compound is indicated in the columnlabeled “L,” and the number of double bonds in each compound isindicated in the column labeled “S.”

FIGS. 4A-4C show the in silico modeling of the binding of compound3-(4-((4-fluoro-4′-methylbiphenyl-2-yl)methoxy)phenyl)propanoic acid(also known as TUG891) to fGPR120. FIG. 4A shows the structure of thebinding compound, FIG. 4B shows a model of the compound binding to thefGPR120, and FIG. 4C shows the putative fGPR120 amino acid residues thatinteract with the binding compound.

FIGS. 5A-5C show the in silico modeling of the binding of compound4-[4-(heptyloxy)phenyl]-4-oxobutanoic acid to fGPR120. FIG. 5A shows thestructure of the binding compound, FIG. 5B shows a model of the compoundbinding to the fGPR120, and FIG. 5C shows the putative fGPR120 aminoacid residues that interact with the binding compound.

FIGS. 6A-6C show the in silico modeling of the binding of compoundeicosapentanoic acid to fGPR120. FIG. 6A Shows the structure of thebinding compound, FIG. 6B shows a model of the compound binding to thefGPR120, and FIG. 6C shows the putative fGPR120 amino acid residues thatinteract with the binding compound.

FIG. 7 shows an overlay of eicosapentanoic acid,3-(4-((4-fluoro-4′-methylbiphenyl-2-yl)methoxy)phenyl)propanoic acid,and 4-[4-(heptyloxy)phenyl]-4-oxobutanoic acid, showing the position ofArg99 of fGPR120 when the receptor is bound by the compounds.

FIGS. 8A and -8B show the in silico modeling of the binding of thecompound oleic acid to fGPR120, wherein a salt bridge is formed betweenthe carboxyl group of oleic acid and Arg99 of fGPR120. FIG. 8A shows thestructure of the binding compound, and FIG. 8B shows a model of thecompound binding to the fGPR120, wherein putative fGPR120 amino acidresidues that interact with the binding compound are shown.

FIGS. 9A-9C show the in silico modeling of the binding of compoundlinoleic acid to fGPR120, wherein a salt bridge is formed between thecarboxyl group of linoleic acid and Arg99 of fGPR120. FIG. 9A shows thestructure of the binding compound, FIG. 9B shows a model of the compoundbinding to the fGPR120, and FIG. 9C shows the putative fGPR120 aminoacid residues that interact with the binding compound.

FIG. 10 show the amino acid sequence of the fGPR120 receptor, identifiedas SEQ ID NO:1.

FIGS. 11A- and 11B show dose response curves for dodecyl dihydrogenphosphate determined in an in vitro cellular assay for activation offGPR120 by the ligand in agonist and PAM modes.

FIGS. 12A and -12B show the dose response curves for the controlsα-linolenic acid and ATP in the in vitro cellular assay for activationof fGPR120, as described by Example 3.

DETAILED DESCRIPTION

To date, there remains a need for a flavor modifier that can increaseand/or enhance the palatability of various cat pet food products. Thepresent application relates to flavor compositions that include at leastone compound. The flavor compositions can be used to increase thepalatability and/or enhance or modify the taste of various pet foodproducts such as a nutritionally-complete pet food. The flavorcompositions can further include combinations of compounds, includingamino acids, nucleotides, and furanones (as described in InternationalApplication Nos. PCT/EP2013/072788 filed Oct. 31, 2013,PCT/EP2013/072789 filed Oct. 31, 2013, PCT/EP2013/072790 filed Oct. 31,2013, and PCT/EP2013/072794 filed Oct. 31, 2013, each of which isincorporated by reference in its entirety), nucleotide derivatives (asdescribed in International Application No. PCT/US15/65046 filed Dec. 10,2015, which is incorporated by reference in its entirety), and/ortransmembrane compounds (as described in International Application No.PCT/US15/65036 filed Dec. 10, 2015, which is incorporated by referencein its entirety) and can be added to pet food products in variousdelivery system formats.

1. Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them.

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.” Still further, the terms“having,” “including,” “containing” and “comprising” are interchangeableand one of skill in the art is cognizant that these terms are open endedterms.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 3 or more than 3 standard deviations,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value.

As used herein, “taste” refers to a sensation caused by activation orinhibition of receptor cells in a subject's taste buds. In certainembodiments, taste can be selected from the group consisting of sweet,sour, salt, bitter, kokumi and umami. In certain embodiments, a taste iselicited in a subject by a “tastant.” In certain embodiments, a tastantis a synthetic tastant. In certain embodiments, the tastant is preparedfrom a natural source.

In certain embodiments, “taste” can include free fatty acid taste. See,e.g., Cartoni et al., J. of Neuroscience, 30(25): 8376-8382 (2010), thecontents of which are incorporated herein by reference. In certainembodiments, free fatty acid taste is a sensation caused by activationor inhibition of receptor cells in a subject's taste buds, for examplethe receptor GPR120, and is separate than the texture and olfactorysensory perceptions of fatty foods, for example, foods with viscous,slippery, oily, mouth-coating or creamy textures.

As used herein, “taste profile” refers to a combination of tastes, suchas, for example, one or more of a sweet, sour, salt, bitter, umami,kokumi and free fatty acid taste. In certain embodiments, a tasteprofile is produced by one or more tastant that is present in acomposition at the same or different concentrations. In certainembodiments, a taste profile refers to the intensity of a taste orcombination of tastes, for example, a sweet, sour, salt, bitter, umami,kokumi and free fatty acid taste, as detected by a subject or any assayknown in the art. In certain embodiments, modifying, changing or varyingthe combination of tastants in a taste profile can change the sensoryexperience of a subject.

As used herein, “flavor” refers to one or more sensory stimuli, such as,for example, one or more of taste (gustatory), smell (olfactory), touch(tactile) and temperature (thermal) stimuli. In certain non-limitingembodiments, the sensory experience of a subject exposed to a flavor canbe classified as a characteristic experience for the particular flavor.For example, a flavor can be identified by the subject as being, but notlimited to, a floral, citrus, berry, nutty, caramel, chocolate, peppery,smoky, cheesy, meaty, etc., flavor. As used herein, a flavor compositioncan be selected from a liquid, solution, dry powder, spray, paste,suspension and any combination thereof. The flavor can be a naturalcomposition, an artificial composition, a nature identical, or anycombination thereof.

As used interchangeably herein, “aroma” and “smell” refer to anolfactory response to a stimulus. For example, and not by way oflimitation, an aroma can be produced by aromatic substances that areperceived by the odor receptors of the olfactory system.

As used herein, “flavor profile” refers to a combination of sensorystimuli, for example, tastes, such as sweet, sour, bitter, salty, umami,kokumi and free fatty acid tastes, and/or olfactory, tactile and/orthermal stimuli. In certain embodiments, the flavor profile comprisesone or more flavors which contribute to the sensory experience of asubject. In certain embodiments, modifying, changing or varying thecombination of stimuli in a flavor profile can change the sensoryexperience of a subject.

As used herein “admixing,” for example, “admixing the flavor compositionor combinations thereof of the present application with a food product,”refers to the process where the flavor composition, or individualcomponents of the flavor composition, is mixed with or added to thecompleted product or mixed with some or all of the components of theproduct during product formation or some combination of these steps.When used in the context of admixing, the term “product” refers to theproduct or any of its components. This admixing step can include aprocess selected from the step of adding the flavor composition to theproduct, spraying the flavor composition on the product, coating theflavor composition on the product, suspending the product in the flavorcomposition, painting the flavor composition on the product, pasting theflavor composition on the product, encapsulating the product with theflavor composition, mixing the flavor composition with the product andany combination thereof. The flavor composition can be a liquid,emulsion, dry powder, spray, paste, suspension and any combinationthereof.

In certain embodiments, the compounds of a flavor composition can begenerated during the processing of a pet food product, e.g.,sterilization, retorting and/or extrusion, from precursor compoundspresent in the pet food product. In a non-limiting example, thecomposition can be a free fatty acid that is liberated from one or moretriglycerides during the processing of a pet food product. In certainembodiments, the free fatty acid is produced during thermal, acid and/orenzymatic treatment of the triglycerides. In certain embodiments, thetriglycerides are present in meat (e.g., chicken, beef, pork, fish,and/or tallow) and/or vegetable (e.g., oils such as vegetable andsunflower oil) sources that are processed to produce the pet food.

As used herein, “ppm” means parts-per-million and is a weight relativeparameter. A part-per-million is a microgram per gram, such that acomponent that is present at 10 ppm is present at 10 micrograms of thespecific component per 1 gram of the aggregate mixture.

As used herein, “palatability” can refer to the overall willingness ofan animal to eat a certain food product. Increasing the “palatability”of a pet food product can lead to an increase in the enjoyment andacceptance of the pet food by the companion animal to ensure the animaleats a “healthy amount” of the pet food. The term “healthy amount” of apet food as used herein refers to an amount that enables the companionanimal to maintain or achieve an intake contributing to its overallgeneral health in terms of micronutrients, macronutrients and calories,such as set out in the “Mars Petcare Essential Nutrient Standards.” Incertain embodiments, “palatability” can mean a relative preference of ananimal for one food product over another. For example, when an animalshows a preference for one of two or more food products, the preferredfood product is more “palatable,” and has “enhanced palatability.” Incertain embodiments, the relative palatability of one food productcompared to one or more other food products can be determined, forexample, in side-by-side, free-choice comparisons, e.g., by relativeconsumption of the food products, or other appropriate measures ofpreference indicative of palatability. Palatability can be determined bya standard testing protocol in which the animal has equal access to bothfood products such as a test called “two-bowl test” or “versus test.”Such preference can arise from any of the animal's senses, but can berelated to, inter alia, taste, aftertaste, smell, mouth feel and/ortexture.

The term “pet food” or “pet food product” means a product or compositionthat is intended for consumption by a companion animal, such as cats,dogs, guinea pigs, rabbits, birds and horses. For example, but not byway of limitation, the companion animal can be a “domestic” cat such asFelis domesticus. In certain embodiments, the companion animal can be a“domestic” dog, e.g., Canis lupus familiaris. A “pet food” or “pet foodproduct” includes any food, feed, snack, food supplement, liquid,beverage, treat, toy (chewable and/or consumable toys), and mealsubstitute or meal replacement.

As used herein “nutritionally-complete” refers to pet food product thatcontains all known required nutrients for the intended recipient of thepet food product, in appropriate amounts and proportions based, forexample, on recommendations of recognized or competent authorities inthe field of companion animal nutrition. Such foods are thereforecapable of serving as a sole source of dietary intake to maintain life,without the addition of supplemental nutritional sources.

As used herein “flavor composition” refers to at least one compound orbiologically acceptable salt thereof that modulates, includingenhancing, multiplying, potentiating, decreasing, suppressing, orinducing, the tastes, smells, flavors and/or textures of a natural orsynthetic tastant, flavoring agent, taste profile, flavor profile and/ortexture profile in an animal or a human. In certain embodiments, theflavor composition comprises a combination of compounds or biologicallyacceptable salts thereof In certain embodiments, the flavor compositionincludes one or more excipients.

As used herein, the terms “modulates” or “modifies” refers an increaseor decrease in the amount, quality or effect of a particular activity ofa receptor and/or an increase or decrease in the expression, activity orfunction of a receptor. “Modulators,” as used herein, refer to anyinhibitory or activating compounds identified using in silico, in vitroand/or in vivo assays for, e.g., agonists, antagonists and theirhomologs, including fragments, variants and mimetics.

“Inhibitors” or “antagonists,” as used herein, refer to modulatingcompounds that reduce, decrease, block, prevent, delay activation,inactivate, desensitize or downregulate biological activity and/orexpression of receptors or pathway of interest.

“Inducers,” “activators” or “agonists,” as used herein, refer tomodulating compounds that increase, induce, stimulate, open, activate,facilitate, enhance activation, sensitize or upregulate a receptor orpathway of interest.

As used herein, the terms “vector” and “expression vector” refer to DNAmolecules that are either linear or circular, into which another DNAsequence fragment of appropriate size can be integrated. Such DNAfragment(s) can include additional segments that provide fortranscription of a gene encoded by the DNA sequence fragment. Theadditional segments can include and are not limited to: promoters,transcription terminators, enhancers, internal ribosome entry sites,untranslated regions, polyadenylation signals, selectable markers,origins of replication and such like. Expression vectors are oftenderived from plasmids, cosmids, viral vectors and yeast artificialchromosomes. Vectors are often recombinant molecules containing DNAsequences from several sources.

The term “operably linked,” when applied to DNA sequences, e.g., in anexpression vector, indicates that the sequences are arranged so thatthey function cooperatively in order to achieve their intended purposes,i.e., a promoter sequence allows for initiation of transcription thatproceeds through a linked coding sequence as far as the terminationsignal.

The term “nucleic acid molecule” and “nucleotide sequence,” as usedherein, refers to a single or double stranded covalently-linked sequenceof nucleotides in which the 3′ and 5′ ends on each nucleotide are joinedby phosphodiester bonds. The nucleic acid molecule can includedeoxyribonucleotide bases or ribonucleotide bases, and can bemanufactured synthetically in vitro or isolated from natural sources.

The terms “polypeptide,” “peptide,” “amino acid sequence” and “protein,”used interchangeably herein, refer to a molecule formed from the linkingof at least two amino acids. The link between one amino acid residue andthe next is an amide bond and is sometimes referred to as a peptidebond. A polypeptide can be obtained by a suitable method known in theart, including isolation from natural sources, expression in arecombinant expression system, chemical synthesis or enzymaticsynthesis. The terms can apply to amino acid polymers in which one ormore amino acid residue is an artificial chemical mimetic of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers and non-naturally occurring amino acidpolymers.

The term “amino acid,” as used herein, refers to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, gamma-carboxyglutamate and O-phosphoserine. Aminoacid analogs and derivatives can refer to compounds that have the samebasic chemical structure as a naturally occurring amino acid, i.e., acarbon that is bound to a hydrogen, a carboxyl group, an amino group andan R group, e.g., homoserine, norleucine, methionine sulfoxide andmethionine methyl sulfonium. Such analogs can have modified R groups(e.g., norleucine) or modified peptide backbones, but retain the samebasic chemical structure as a naturally occurring amino acid. Amino acidmimetics means chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunctions in a manner similar to a naturally occurring amino acid.

The terms “isolated” or “purified,” used interchangeably herein, refersto a nucleic acid, a polypeptide, or other biological moiety that isremoved from components with which it is naturally associated. The term“isolated” can refer to a polypeptide that is separate and discrete fromthe whole organism with which the molecule is found in nature or ispresent in the substantial absence of other biological macromolecules ofthe same type. The term “isolated” with respect to a polynucleotide canrefer to a nucleic acid molecule devoid, in whole or part, of sequencesnormally associated with it in nature; or a sequence, as it exists innature, but having heterologous sequences in association therewith; or amolecule disassociated from the chromosome.

As used herein, the term “recombinant” can be used to describe a nucleicacid molecule and refers to a polynucleotide of genomic, RNA, DNA, cDNA,viral, semisynthetic or synthetic origin which, by virtue of its originor manipulation is not associated with all or a portion ofpolynucleotide with which it is associated in nature.

The term “fusion,” as used herein, refers to joining of differentpeptide or protein segments by genetic or chemical methods wherein thejoined ends of peptide or protein segments may be directly adjacent toeach other or may be separated by linker or spacer moieties such asamino acid residues or other linking groups.

2. Fatty Acid Receptor

The presently disclosed subject matter provides fatty acid receptors foruse in the disclosed methods. The fatty acid receptors of the presentdisclosure can include mammalian fatty acid receptors such as, but notlimited to, feline, canine and human fatty acid receptors.

In certain non-limiting embodiments, the fatty acid receptor of thepresent disclosure is encoded by a nucleic acid as described byInternational Publication No. WO 2014/199114, published Dec. 18, 2014,which is incorporated by reference in its entirety herein. In certainnon-limiting embodiments, the fatty acid receptor of the presentdisclosure comprises an amino acid sequence as described byInternational Publication No. WO 2014/199114.

In certain non-limiting embodiments, the fatty acid receptor comprises afeline, canine or human fatty acid receptor nucleotide sequence asdescribed by International Publication No. WO 2014/199114, publishedDec. 18, 2014.

In certain non-limiting embodiments, the fatty acid receptor comprises afeline, canine or human fatty acid receptor amino acid sequence asdescribed by International Publication No. WO 2014/199114, publishedDec. 18, 2014.

In certain embodiments, the fatty acid receptor for use in the presentlydisclosed subject matter can include a receptor comprising a nucleotidesequence having at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identity to a feline, canine or human fattyacid receptor nucleotide sequence.

In certain embodiments, the fatty acid receptor for use in the presentlydisclosed subject matter can include a receptor comprising an amino acidsequence having at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identity to a feline, canine or human fattyacid receptor amino acid sequence.

In certain embodiments, the disclosed subject matter provides for theuse of an isolated or purified fatty acid receptor and/or variants andfragments thereof. The disclosed subject matter also encompasses the useof sequence variants. In certain embodiments, variation can occur ineither or both the coding and non-coding regions of a nucleotidesequence of a fatty acid receptor. Variants can include a substantiallyhomologous protein encoded by the same genetic locus in an organism,i.e., an allelic variant. Variants also encompass proteins derived fromother genetic loci in an organism, e.g., feline, but having substantialhomology to the fatty acid receptor, i.e., a homolog. Variants can alsoinclude proteins substantially homologous to the fatty acid receptor butderived from another organism, i.e., an ortholog. Variants also includeproteins that are substantially homologous to the fatty acid receptorthat are produced by chemical synthesis. Variants also include proteinsthat are substantially homologous to the fatty acid receptor that areproduced by recombinant methods.

The disclosed subject matter also provides for fusion proteins thatcomprise a fatty acid receptor, or fragment thereof. In certainembodiments, a fusion protein of the present disclosure can include adetectable marker, a functional group such as a carrier, a label, astabilizing sequence or a mechanism by which fatty acid receptor agonistbinding can be detected. Non-limiting embodiments of a label include aFLAG tag, a His tag, a MYC tag, a maltose binding protein and othersknown in the art. The presently disclosed subject matter also providesnucleic acids encoding such fusion proteins, vectors containing fusionprotein-encoding nucleic acids and host cells comprising such nucleicacids or vectors. In certain embodiments, fusions can be made at theamino terminus (N-terminus) of a fatty acid receptor or at the carboxyterminus (C-terminus) of a fatty acid receptor.

In certain embodiments, the fatty acid receptors disclosed herein cancontain additional amino acids at the N-terminus and/or at theC-terminus end of the sequences, e.g., when used in the methods of thedisclosed subject matter. In certain embodiments, the additional aminoacids can assist with immobilizing the polypeptide for screeningpurposes, or allow the polypeptide to be part of a fusion protein, asdisclosed above, for ease of detection of biological activity.

3. Fatty Acid Receptor Binding Compounds

The present disclosure relates to flavor compositions comprising atleast one compound that can modulate the activity of a fatty acidreceptor, for example, a GPR120 receptor. In certain embodiments, thecompositions comprise a free fatty acid. The compounds disclosed hereinwere identified through an in vitro assay wherein the ability of thecompounds to activate a feline GPR120 receptor (fGPR120) expressed bycells in culture was determined, and/or an in silico assay, wherein thecompounds' ability to bind to fGPR120 was determined in silico. Theflavor compositions can be used to enhance or modify the palatability,taste or flavor of pet food products. The flavor compositions caninclude combinations of compounds, for example, combinations of one ormore compounds and/or one or more amino acids and/or one or morenucleotides and/or one or more furanones as described herein and inInternational Application Nos. PCT/EP2013/072788 filed Oct. 31, 2013,PCT/EP2013/072789 filed Oct. 31, 2013, PCT/EP2013/072790 filed Oct. 31,2013, PCT/EP2013/072794 filed Oct. 31, 2013, each of which isincorporated by reference herein in its entirety; and/or one or morenucleotide derivatives as described herein and in InternationalApplication No. PCT/US15/65046 filed Dec. 10, 2015, which isincorporated by reference herein in its entirety; and/or one or moretransmembrane compounds, as described herein and in InternationalApplication No. PCT/US15/65036 filed Dec. 10, 2015, which isincorporated by reference herein in its entirety, and can be added topet food product compositions in various delivery system formats.

In certain embodiments, at least one fatty acid receptor modulatingcompound is selected from the following compounds:

(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoic acid (also known asArachidonic Acid);

(5Z,8Z)-icosa-5,8-dienoic acid;

4-[4-(heptyloxy)phenyl]-4-oxobutanoic acid;

(11Z)-octadec-11-enoic acid (also known as cis-Vaccenic acid);

(9E)-hexadec-9-enoic acid (also known as Palmitelaidic acid);

tridec-12-enoic acid (also known as 12-Tridecenoic acid);

S-Farnesyl Thioacetic Acid;

(10Z)-pentadec-10-enoic acid (also known as (10Z)-10-Pentadecenoicacid);

10(E),12(Z)-Conjugated Linoleic Acid (also known as(10Z,12Z)-10,12-Octadecadienoic acid);

(10Z,13Z)-nonadeca-10,13-dienoic acid;

(9Z,11E)-octadeca-9,11-dienoic acid;

cis-7-Hexadecenoic Acid;

dodecanoic acid (also known as Lauric acid);

(9Z)-tetradec-9-enoic acid (also known as Myristoleic acid);

(11Z,14Z,17Z)-icosa-11,14,17-trienoic acid (also known asDihomo-α-linolenic acid (20:3(n-3)));

(6Z,9Z,12Z)-octadeca-6,9,12-trienoic acid (also known as γ-Linolenicacid);

(11Z,14Z)-icosa-11,14-dienoic acid (also known as Dihomolinoleic acid(20:2(n-6)));

(9Z)-hexadec-9-enoic acid (also known as palmitoleate, (Z)-Palmitoleicacid);

12-methoxydodecanoic acid;

(8Z,11Z,14Z)-icosa-8,11,14-trienoic acid;

(9Z,12Z)-octadeca-9,12-dienoic acid (also known as Linoleic acid);

(10Z)-heptadec-10-enoic acid;

Pinolenic Acid;

(9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid (also known as α-Linolenicacid)1;

tridecanoic acid (also known as Tridecylic acid);

tetradecanoic acid (also known as Myristic acid);

(9Z)-octadec-9-enoic acid (also known as Oleic acid);

GW 9508 (also known as4-[[(3-Phenoxyphenyl)methyl]amino]benzenepropanoic acid);

(5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenoic acid (also known asEicosapentanoic acid);

3-(4-((4-fluoro-4′-methylbiphenyl-2-yl)methoxy)phenyl)propanoic acid(also known as TUG 891 and4-[(4-Fluoro-4′-methyl[1,1′-biphenyl]-2-yl)methoxy]-benzenepropanoicacid);

(10E)-pentadec-10-enoic acid;

(9E)-tetradec-9-enoic acid (also known as Myristoleate);

(6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraenoic acid;

(5Z,8Z,11Z)-icosa-5,8,11-trienoic acid (also known as Mead acid); and

Dodecyl dihydrogen phosphate.

The present disclosure also provides for salts, stereoisomers andcomestible forms of the compounds described herein.

In certain embodiments, the compound is a saturated fatty acid.

In certain embodiments, the compound is an unsaturated orpolyunsaturated fatty acid.

In certain embodiments, the compound comprises the structure R—X,wherein

X is a carboxyl group or a bioisosteric replacement for a caboxylatesuch as, but not limited to, a phosphate; boronic acid; phosphoester;phosphodiester; phosphinic acid; sulfonamide; malonic acid;2-sulfoacetic acid; sulfonic acid; hydroxamic acid; tetrazole;hydroxylurea; phosphoramide, C(O)OCH₂CH(OH)OP(O)(OR₁)(OR₂) where R₁ andR₂ are each independently selected from H, lower alkyl (C₁-C₆), and Ph;or any other bioisosteric replacement for a caboxylate group known inthe art; and

R is a hydrocarbon chain having a carbon length of between about C₂ andabout C₄₀, or between about C₄ and about C₃₅, or between about C₆ andabout C₃₀, or between about C₈ and about C₂₈, or between about C₁₀ andabout C₂₆, or between about C₁₂ and about C₂₄, or between about C₁₄ andabout C₂₂, or between about C₁₆ and about C₂₀. In certain embodiments,the hydrocarbon chain has a carbon length of between about C₁₄ and aboutC₂₂.

In certain embodiments, the hydrocarbon chain may be unsubstituted orsubstituted with one or more of H, CH₃, C₁-C₂₀, COOH, SO₃H, CHO, C(O)R₁,C═CR₁R₂, C═NOH, OP(O)(OR₁)(OR₂), OR₁, SR₁, CN, COOR₁, CONR₁R₂, NO₂,NHOH, F, Cl, Br, I, and NR₁R₂, where R₁, R₂ are each independentlyselected from H and branched or unbranched lower alkyl (e.g., C₁-C₆).

In certain embodiments the hydrocarbon chain comprises zero to eightdouble bonds, for example, positioned alternately with single bondsalong the hydrocarbon chain. In certain embodiments, the compound is an(E) isomer. In certain embodiments, the compound is a (Z) isomer. Incertain embodiments, the double bonds may optionally be substituted withone or more of hydrogen, methyl, or lower alkyl groups, for example, analkyl having a carbon length of between about C₁ and C₁₂.

In certain embodiments, one or more of the carbons in the hydrocarbonchain is substituted by an oxygen, sulfur, nitrogen, ketone, sulfone,sulfoxide, amide, urea, or sulfonamide.

In certain embodiments, the compounds of the present disclosure compriseone or more negatively charged groups attached to a hydrocarbon chain.In certain embodiments, the negatively charged group comprises, forexample, a carboxyl group, or a bioisosteric replacement for acaboxylate such as, but not limited to, a phosphate, boronic acid,phosphoester, phosphodiester, phosphinic acid, sulfonamide, malonicacid, 2-sulfoacetic acid, sulfonic acid, hydroxamic acid, tetrazole,hydroxylurea, phosphoramide, or any other bioisosteric replacement for acaboxylate known to a practitioner in the art.

In certain embodiments the compounds comprise a fatty acid. In certainembodiments, the fatty acid does not comprise a fatty acid ester, forexample, a methyl ester or ethyl ester.

In certain embodiments the compounds comprise a carboxyl group thatinteracts with arginine at position 99 of GPR120 (i.e., Arg99). Incertain embodiments, the carboxyl group forms a salt bridge with Arg99.In certain embodiments, the hydrocarbon chain of the compound formshydrophobic interactions with other GPR120 amino acid residues presentin the GPR120 fatty acid binding domain.

In certain embodiments the compound of the present disclosure comprisesa compound of Formula I, having the following structure:

wherein R is a hydrocarbon chain, for example, a substituted orunsubstituted hydrocarbon chain having a carbon length of between aboutC₁ and about C₄₀, or between about C₄ and about C₃₅, or between about C₆and about C₃₀, or between about C₈ and about C₂₈, or between about C₁₀and about C₂₆, or between about C₁₂ and about C₂₄, or between about C₁₄and about C₂₂, or between about C₁₆ and about C₂₀. In certainembodiments, the hydrocarbon chain has a carbon length of between aboutC₁₄ and about C₂₂. Substitutions may include, but are not limited to,those described above, for example, the hydrocarbon chain may besubstituted with one or more of H, CH₃, C₁-C₂₀, COOH, SO₃H, CHO, C(O)R₁,C═CR₁R₂, C═NOH, OP(O)(OR₁)(OR₂), OR₁, SR₁, CN, COOR₁, CONR₁R₂, NO₂,NHOH, F, Cl, Br, I, and NR₁R₂, where R₁, R₂ are each independentlyselected from H or branched or unbranched lower alkyl (e.g., C₁-C₆).

In certain embodiments the compound of the present disclosure comprisesa compound of Formula II, having the following structure:

wherein

X is (C)R₁R₂, O, S, C(O), or a chemical bond;

X₁ is (C)R₁R₂, O, S, or N(R₁);

Y is COOH, SO₃H, —OP(O)(OH)₂, or Tetrazole;

W is halogen, COOH, COOMe, CN, N(R₁)(R₂), CHO, CONR₁R₂, Aryl, OH, H,—S-Aryl, —O-Aryl, —N-Aryl, CF₃, OCH₃, CH₃, NO₂, or OEt;

Z₁ and Z₂ are each independently selected from halogen, COOH, COOMe, CN,N(R₁)(R₂), CHO, CONR₁R₂, Aryl, OH, H, —S-Aryl, —O-Aryl, —N-Aryl, CF₃,OCH₃, CH₃, NO₂, and OEt;

R₁ and R₂ are each independently selected from H and branched orunbranched C₁-C₆ lower alkyl;

n₁ is 0-4;

n₂ is 0-2; and

n₃ is 0-4.

In certain embodiments the compound of the present disclosure comprisesa compound of Formula III, having the following structure:

wherein

X is (C)R₁R₂, O, S, C(O), or a chemical bond;

X₁ is (C)R₁R₂, O, S, or N(R₁);

Y is COOH, SO₃H, —OP(O)(OH)₂, or Tetrazole;

W is halogen, COOH, COOMe, CN, N(R₁)(R₂), CHO, CONR₁R₂, Aryl, OH, H,—S-Aryl, —O-Aryl, —N-Aryl, CF₃, OCH₃, CH₃, NO₂, or OEt;

R is CH₃, or (R₄)(R₅)X₂;

R₁ and R₂ are each independently selected from H and branched orunbranched C₁-C₆ lower alkyl;

R₄ and R₅ are each independently selected from H, aryl, and branched orunbranched lower alkyl (C₁-C₁₀);

n₁ is 0-4;

n₂ is 0-2;

n₃ is 1-12 (branched or unbranched C₂-C₂₀); and

X₂ is C or N.

In certain embodiments, the fatty acid receptor modulators of thepresent disclosure comprise a salt of the fatty acid receptor modulator,for example, but not limited to, an acetate salt or a formate salt. Incertain embodiments, the fatty acid receptor modulator salt comprises ananion (−) (for example, but not limited to, Cl⁻, O²⁻, CO₃ ²⁻, HCO₃ ⁻,OH⁻, NO₃ ⁻, PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, HCOO⁻ and C₂O₄ ²⁻) bonded via anionic bond with a cation (+) (for example, but not limited to, Al³⁺,Ca²⁺, Na⁺, K⁺, Cu²⁺, H⁺, Fe³⁺, Mg²⁺, NH₄ ⁺ and H₃O⁺). In otherembodiments, the fatty acid receptor agonist salt comprises a cation (+)bonded via an ionic bond with an anion (−). In certain embodiments, thecompounds of the present disclosure comprise a sodium salt or potassiumsalt of the compound.

In certain embodiments, the fatty acid receptor modulators of thepresent application are identified through in silico modeling of a fattyacid receptor (such as a GPR120 receptor), e.g., a feline or a caninefatty acid receptor, wherein the fatty acid receptor modulatingcompounds of the present application comprise a structure that fitswithin a binding site of the fatty acid receptor. In certainembodiments, the in silico method comprises the in silico methodsdescribed herein and in the Examples section of the present application.

In certain embodiments, the fatty acid receptor modulators of thepresent application are identified through an in vitro method, whereinthe fatty acid receptor agonist compounds activate and/or modulate afatty acid receptor, disclosed herein, expressed by cells in vitro. Incertain embodiments, the in vitro method comprises the in vitro methodsdescribed herein and in the Examples section of the present application.

In certain embodiments, the compounds are comprised in a flavorcomposition without other palatability enhancing agents. In certainembodiments, the compounds are comprised in one or more flavorcompositions with one or more additional palatability enhancing agents,for example, nucleotides, nucleotide derivatives, amino acids, furanonesand transmembrane compounds described herein which activate differentactive sites on different receptors (e.g., an umami receptor).

4. Methods for Identifying Fatty Acid Receptor Modulating Compounds

The present disclosure further provides methods for identifyingcompounds that modulate the activity and/or expression of a fatty acidreceptor. For example, and not by way of limitation, the modulator canbe an agonist or an antagonist. The presently disclosed subject matterprovides in silico and in vitro methods for identifying compounds thatmodulate the activity and/or expression of a fatty acid receptor,disclosed above.

4.1 In Silico Methods

The presently disclosed subject matter further provides in silicomethods for identifying compounds that can potentially interact with afatty acid receptor and/or modulate the activity and/or expression of afatty acid receptor, for example, a feline, canine or human fatty acidreceptor.

In certain embodiments, the method can include predicting thethree-dimensional structure (3D) of a fatty acid receptor and screeningthe predicted 3D structure with putative fatty acid receptor modulatingcompounds (i.e., test compounds). The method can further includepredicting whether the putative compound would interact with the bindingsite of the receptor by analyzing the potential interactions with theputative compound and the amino acids of the receptor. The method canfurther include identifying a test compound that can bind to and/ormodulate the biological activity of the fatty acid receptor bydetermining whether the 3D structure of the compound fits within thebinding site of the 3D structure of the receptor.

In certain embodiments, the fatty acid receptor for use in the disclosedmethod can have an amino acid or nucleotide sequence as described byInternational Publication No. WO 2014/199114, published Dec. 18, 2014,or a fragment or variant thereof.

Non-limiting examples of compounds (e.g., potential fatty acid receptormodulators) that can be tested using the disclosed methods include anysmall chemical compound, or any biological entity, such as peptides,salts, amino acids and fatty acid compounds known in the art. In certainembodiments, the test compound can be a small chemical molecule.

In certain embodiments, structural models of a fatty acid receptor canbe built using crystal structures of other GPCRs as templates forhomology modeling. For example, and not by way of limitation, structuralmodels can be generated using the crystal structures of Group A GPCRs.In certain embodiments, a structural model of a fatty acid receptor canbe based on a known or a combination of known crystal structures ofGPCRs. (See, e.g., Rasmussen, S. G. et al., (2011) Nature 477: 549-555;Wu, B. et al., (2010) Science 330: 1066-1071; and Wu, H. et al., (2012)Nature 485: 327-332, each of which is incorporated by reference in itsentirety herein). For example, and not by way of limitation, astructural model of the transmembrane domain of a fatty acid receptorcan be generated based on the crystal structure having the protein database (PDB) ID No. 4DJH, 3OE6, and/or 3SN6. FIGS. 4A-4C, 5A-5C, 6A-6C,8A, 8B and 9A-9C depict structural models of fatty acid receptors thatcan be used in the disclosed in silico methods. Any suitable modelingsoftware known in the art can be used. In certain embodiments, theModeler software package (Accelrys, BIOVIA, Dassault Systèmes) can beused to generate the three-dimensional protein structure.

In certain embodiments, the in silico methods of identifying a compoundthat binds to a fatty acid receptor comprises determining whether a testcompound interacts with one or more amino acids of a fatty acid receptorinteracting domain, as described herein.

Compounds that are identified by the disclosed in silico methods can befurther tested using the in vitro methods disclosed herein.

4.2 Fatty Acid Receptor Binding Site

The present application provides for methods of screening for compoundsthat modulate the activity of a fatty acid receptor, for example, afeline, canine or human fatty acid receptor, wherein the compoundsinteract with one or more amino acids of the fatty acid receptor. Incertain embodiments, the binding site of a fatty acid receptor comprisesamino acids within the transmembrane domain, for example,7-transmembrane (7TM) domain, of the receptor, and can be identified bygenerating an interaction map of the receptor using in silico modeling,as described herein. In one non-limiting example, the presence of anamino acid in the transmembrane interaction map means that the residueis in the vicinity of the ligand binding environment, and interacts withthe ligand.

In certain embodiments, the interaction between a compound and one ormore amino acids of the fatty acid receptors described herein cancomprises one or more hydrogen bond, covalent bond, non-covalent bond,salt bridge, physical interaction, and combinations thereof. Theinteractions can also be any interaction characteristic of a ligandreceptor interaction known in the art. Such interactions can be deterwined by, for example, site directed mutagenesis, x-ray crystallography,x-ray or other spectroscopic methods, Nuclear Magnetic Resonance (NMR),cross-linking assessment, mass spectroscopy or electrophoresis,cryo-microscopy, displacement assays based on known agonists, structuraldetermination and combinations thereof. In certain embodiments, theinteractions are determined in silico, for example, by theoretical meanssuch as docking a compound into a feline or canine fatty acid receptorbinding pocket as described herein, for example, using moleculardocking, molecular modeling, molecular simulation, or other means knownto persons of ordinary skill in the art.

In certain embodiments, the interaction is a hydrogen bond interaction.

In certain embodiments, the interaction is a hydrophobic interaction.

In certain embodiments, the compounds identified according to themethods described herein that modulate the activity of a fatty acidreceptor interact with one or more amino acids in a transmembrane domainof the fatty acid receptor, for example, a seven transmembrane domain(7TM). In certain embodiments, the amino acids that the compoundsinteract with comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 or more of PHE88, VAL95, VAL98 andARG99 on Helix 2; PHE115, MET118, SER119, GLY122, SER123 on Helix 3;TRP207, PHE211, VAL212, ASN215 on Helix 5; TRP277, ILE280, ILE281,ILE284 on Helix 6; and TRP299, PHE303, PHE304, VAL307, THR310, PHE311 onHelix 7 of a fatty acid receptor, for example, a fatty acid receptorcomprising a feline fatty acid receptor, or the corresponding aminoacids of a canine fatty acid receptor or a human fatty acid receptor.

In certain embodiments, the methods for identifying a composition thatmodulates the activity of a feline fatty acid receptor comprises (a)contacting a test agent with a fatty acid receptor, for example, afeline fatty acid receptor comprising an amino acid sequence of SEQ IDNO: 1, (b) detecting an interaction between the test agent and one ormore amino acids in an interacting site of the fatty acid receptorselected from the group consisting of PHE88, VAL95, VAL98 and ARG99 onHelix 2; PHE115, MET118, SER119, GLY122, SER123 on Helix 3; TRP207,PHE211, VAL212, ASN215 on Helix 5; TRP277, ILE280, ILE281, ILE284 onHelix 6; and TRP299, PHE303, PHE304, VAL307, THR310, PHE311 on Helix 7;and combinations thereof, and (c) selecting as the composition, a testagent that interacts with one or more of the amino acids.

In certain embodiments, the method further comprises determining theactivity of the fatty acid receptor after step (a), and selecting as thecomposition, a test agent that increases the activity of the fatty acidreceptor.

In certain embodiments, the method further comprises contacting thefatty acid receptor with a ligand, for example an agonist, and selectingas the composition, a test agent that increases or enhances the agonisesability to activate the fatty acid receptor.

4.3 In Vitro Methods

The presently disclosed subject matter further provides in vitro methodsfor identifying compounds that can modulate the activity and/orexpression of a fatty acid receptor.

The fatty acid receptors for use in the presently disclosed methods caninclude isolated or recombinant fatty acid receptors or cells expressinga fatty acid receptor, disclosed herein. In certain embodiments, thefatty acid receptor for use in the disclosed methods can have an aminoacid or nucleotide sequence as described by International PublicationNo. WO 2014/199114, published Dec. 18, 2014, or a fragment or variantthereof.

In certain embodiments, the method for identifying compounds thatmodulate the activity and/or expression of a fatty acid receptorcomprises measuring the biological activity of a fatty acid receptor inthe absence and/or presence of a test compound. In certain embodiments,the method can include measuring the biological activity of a fatty acidreceptor in the presence of varying concentrations of the test compound.The method can further include identifying the test compounds thatresult in a modulation of the activity and/or expression of the fattyacid receptor compared to the activity and/or expression of the fattyacid receptor in the absence of the test compound.

In certain embodiments, the compounds identified according to themethods described herein increase the biological activity of a fattyacid receptor by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more,compared to the biological activity of the fatty acid receptor when thecompound is not present. In certain embodiments, the compoundsidentified according to the methods described herein increase thebiological activity of a fatty acid receptor by at least about 30%compared to the biological activity of the fatty acid receptor when thecompound is not present.

In certain embodiments, the method can further include analyzing two ormore, three or more or four or more test compounds in combination. Incertain embodiments, the two or more, three or more or four or more testcompounds can be from different classes of compounds, e.g., amino acidsand small chemical compounds. For example, and not by way of limitation,the method can include analyzing the effect of one or more smallchemical test compounds on the biological activity and/or expression ofa fatty acid receptor in the presence of one or more amino acid testcompounds. In certain embodiments, the method for identifying acompound's effect on the activity and/or expression of a fatty acidreceptor comprises analyzing the effect of a test compound on thebiological activity and/or expression of a fatty acid receptor in thepresence of one or more nucleotide or nucleotide derivative testcompounds.

In certain embodiments, the method for identifying compounds thatmodulate the activity and/or expression of a fatty acid receptorcomprises determining whether a compound modulates the receptordirectly, for example, as an agonist or antagonist. In certainembodiments, the method comprises determining whether a compoundindirectly modulates the activity of the receptor (e.g., as anallosteric modulator), for example, by enhancing or decreasing theeffect of other compounds on activating or inhibiting receptor activity.

In certain embodiments, the method for identifying compounds thatmodulate the activity and/or expression of a fatty acid receptorcomprises expressing a fatty acid receptor in a cell line and measuringthe biological activity of the receptor in the presence and/or absenceof a test compound. The method can further comprise identifying testcompounds that modulate the activity of the receptor by determining ifthere is a difference in receptor activation in the presence of a testcompound compared to the activity of the receptor in the absence of thetest compound. In certain embodiments, the selectivity of the putativefatty acid receptor modulator can be evaluated by comparing its effectson other GPCRs or taste receptors, e.g., umami, CaSR, T1R, etc.receptors.

Activation of the receptor in the disclosed methods can be detectedthrough the use of a labelling compound and/or agent. In certainembodiments, the activity of the fatty acid receptor can be determinedby the detection of secondary messengers such as, but not limited to,cAMP, cGMP, IP3, DAG or calcium. In certain embodiments, the activity ofthe fatty acid receptor can be determined by the detection of theintracellular calcium levels. Monitoring can be by way of luminescenceor fluorescence detection, such as by a calcium sensitive fluorescentdye. In certain embodiments, the intracellular calcium levels can bedetermined using a cellular dye, e.g., a fluorescent calcium indicatorsuch as Calcium 4. In certain embodiments, the intracellular calciumlevels can be determined by measuring the level of calcium binding to acalcium-binding protein, for example, calmodulin. Alternatively and/oradditionally, activity of the fatty acid receptor can be determined bydetection of the phosphorylation, transcript levels and/or proteinlevels of one or more downstream protein targets of the fatty acidreceptor.

The cell line used in the disclosed methods can include any cell typethat is capable of expressing a fatty acid receptor. Non-limitingexamples of cells that can be used in the disclosed methods include HeLacells, Chinese hamster ovary cells (CHO cells), African green monkeykidney cells (COS cells), Xenopus oocytes, HEK-293 cells and murine 3T3fibroblasts. In certain embodiments, the method can include expressing afatty acid receptor in CHO-K1 cells. In certain embodiments, the methodcan include expressing a fatty acid receptor in HEK-293 cells. Incertain embodiments, the method can include expressing a fatty acidreceptor in COS cells. In certain embodiments, the cells constitutivelyexpress the fatty acid receptor. In another embodiment, expression ofthe fatty acid receptor by the cells is inducible.

In certain embodiments, the cell expresses a calcium-bindingphotoprotein, wherein the photoprotein luminesces upon binding calcium.In certain embodiments, the calcium binding photoprotein comprises theprotein clytin. In certain embodiments the clytin is a recombinantclytin. In certain embodiments, the clytin comprises an isolated clytin,for example, a clytin isolated from Clytia gregarium. In certainembodiments, the calcium-binding photoprotein comprises the proteinaequorin, for example, a recombinant aequorin or an isolated aequorin,such as an aequorin isolated from Aequorea victoria. In certainembodiments, the calcium-binding photoprotein comprises the proteinobelin, for example, a recombinant obelin or an isolated obelin, such asan obelin isolated from Obelia longissima.

In certain embodiments, expression of a fatty acid receptor in a cellcan be performed by introducing a nucleic acid encoding a fatty acidreceptor into the cell. For example, and not by way of limitation, anucleic acid having the nucleotide sequence set forth in InternationalPublication No. WO 2014/199114, published Dec. 18, 2014, or a fragmentthereof, can be introduced into a cell. In certain embodiments, theintroduction of a nucleic acid into a cell can be carried out by anymethod known in the art, including but not limited to transfection,electroporation, microinjection, infection with a viral or bacteriophagevector containing the nucleic acid sequences, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer,spheroplast fusion, etc. Numerous techniques are known in the art forthe introduction of foreign genes into cells (see, e.g., Loeffler andBehr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol.217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92 (1985), thedisclosures of which are hereby incorporated by reference in theirentireties) and can be used in accordance with the disclosed subjectmatter. In certain embodiments, the technique can provide for stabletransfer of nucleic acid to the cell, so that the nucleic acid isexpressible by the cell and inheritable and expressible by its progeny.In certain embodiments, the technique can provide for a transienttransfer of the nucleic acid to the cell, so that the nucleic acid isexpressible by the cell, wherein heritability and expressibilitydecrease in subsequent generations of the cell's progeny.

In certain embodiments, the method can include identifying compoundsthat bind to a fatty acid receptor. The method can comprise contacting afatty acid receptor with a test compound and measuring binding betweenthe compound and the fatty acid receptor. For example, and not by way oflimitation, the methods can include providing an isolated or purifiedfatty acid receptor in a cell-free system, and contacting the receptorwith a test compound in the cell-free system to determine if the testcompound binds to the fatty acid receptor. In certain embodiments, themethod can comprise contacting a fatty acid receptor expressed on thesurface of a cell with a test compound and detecting binding of the testcompound to the fatty acid receptor. The binding can be measureddirectly, e.g., by using a labeled test compound, or can be measuredindirectly. In certain embodiments, the detection comprises detecting aphysiological event in the cell caused by the binding of the compound tothe fatty acid receptor, e.g., an increase in the intracellular calciumlevels. For example, and not by way of limitation, detection can beperformed by way of fluorescence detection, such as a calcium sensitivefluorescent dye, by detection of luminescence, or any other method ofdetection known in the art.

In certain non-limiting embodiments, the in vitro assay comprises cellsexpressing a fatty acid receptor that is native to the cells. Examplesof such cells expressing a native fatty acid receptor include, forexample but not limited to, dog (canine) and/or cat (feline) taste cells(e.g., primary taste receptor cells). In certain embodiments, the dogand/or cat taste cells expressing a fatty acid receptor are isolatedfrom a dog and/or cat and cultured in vitro. In certain embodiments, thetaste receptor cells can be immortalized, for example, such that thecells isolated from a dog and/or cat can be propagated in culture.

In certain embodiments, expression of a fatty acid receptor in a cellcan be induced through gene editing, for example, through use of theCRISPR gene editing system to incorporate a fatty acid receptor geneinto the genome of a cell, or to edit or modify a fatty acid receptorgene native to the cell.

In certain embodiments, the in vitro methods of identifying a compoundthat binds to a fatty acid receptor comprises determining whether a testcompound interacts with one or more amino acids of a fatty acid receptorinteracting domain, as described herein.

In certain embodiments, compounds identified as modulators of a fattyacid receptor can be further tested in other analytical methodsincluding, but not limited to, in vivo assays, to confirm or quantitatetheir modulating activity.

In certain embodiments, methods described herein can comprisedetermining whether the fatty acid receptor modulator is a fatty acidtaste enhancing compound, e.g., a fatty acid receptor agonist.

In certain embodiments, the methods of identifying a fatty acid receptormodulator can comprise comparing the effect of a test compound to afatty acid receptor agonist. For example, a test compound that increasesthe activity of the receptor compared to the activity of the receptorwhen contacted with a fatty acid receptor agonist can be selected as afatty acid receptor modulating compound (e.g., as an agonist).

In certain embodiments, the methods of identifying a fatty acid receptormodulator can comprise determining whether a test compound modulates theactivity of the receptor when the receptor is contacted with an agonist,or whether the test compound can modulate the activity of a positiveallosteric modulator (PAM). Test compounds that increase or decrease theeffect of said agonist or PAM on the receptor can be selected as a fattyacid receptor modulating compound (e.g., as an allosteric modulator).

5. Flavor Compositions

In certain embodiments, the flavor compositions of the presentdisclosure can be used to increase the palatability of pet foodproducts, such as cat food products. The flavor compositions can includecombinations of compounds, and can be added to the pet food product invarious delivery systems.

In certain embodiments, the present disclosure relates to methods formodulating the fatty acid taste (for example, the activity of a fattyacid receptor, such as GPR120, including fGPR120) and/or thepalatability of a pet food product comprising: a) providing at least onepet food product, or a precursor thereof, and b) combining the pet foodproduct, or precursor thereof, with at least a fatty acid tastemodulating amount of at least one flavor composition, for example,comprising one or more compounds, or a comestibly acceptable saltthereof, so as to form an enhanced pet food product.

In certain embodiments, the flavor compositions of the presentdisclosure can enhance the activity of a GPR120 receptor and/orpalatability of a pet food product, such as, for example, a pet foodproduct including wet pet food products, dry pet food products, moistpet food products, pet beverage products and/or snack pet food products.

In certain embodiments, one or more of the flavor compositions of thepresent disclosure can be added to a pet food product, in an amounteffective to modify, enhance or otherwise alter a taste or taste profileof the pet food product. The modification can include, for example, anincrease or enhancement in the palatability of the pet food product, asdetermined by animals, e.g., cats and/or dogs, or in the case offormulation testing, as determined by a panel of animal taste testers,e.g., cats and/or dogs, via procedures known in the art.

In certain embodiments of the present disclosure, a pet food product canbe produced that contains a sufficient amount of at least one flavorcomposition described herein, for example, comprising a compound, toproduce a pet food product having the desired taste, e.g., fatty acidtaste.

In certain embodiments of the present disclosure, a pet food product canbe produced that contains a sufficient amount of a flavor compositioncomprising at least one, two, three, four, five, six or more compounds.

In certain embodiments, a GPR120 modulating amount of one or more of theflavor compositions of the present disclosure can be added to the petfood product, so that the pet food product has an increased palatabilityas compared to a pet food product prepared without the flavorcomposition, as determined by animals, e.g., cats and/or dogs, or in thecase of formulation testing, as determined by a panel of animal tastetesters, via procedures known in the art.

In certain embodiments of the present disclosure, the flavor compositionis added to a pet food product in an amount effective to increase,enhance and/or modify the palatability of the pet food product.

The concentration of flavor composition admixed with a pet food productto modulate and/or improve the palatability of the pet food product canvary depending on variables, such as, for example, the specific type ofpet food product, what fatty acid compounds are already present in thepet food product and the concentrations thereof, and the enhancer effectof the particular flavor composition on such fatty acid compounds.

A broad range of concentrations of the flavor compositions can beemployed to provide such palatability modification. In certainembodiments of the present application, the flavor composition isadmixed with a pet food product wherein the flavor composition ispresent in an amount of from about 0.01 ppm to about 1,000 ppm. Forexample, but not by way of limitation, the flavor composition can bepresent in the amount from about 0.01 ppm to about 750 ppm, from about0.01 ppm to about 500 ppm, from about 0.01 ppm to about 250 ppm, fromabout 0.01 ppm to about 150 ppm, from about 0.01 ppm to about 100 ppm,from about 0.01 ppm to about 75 ppm, from about 0.01 ppm to about 50ppm, from about 0.01 ppm to about 25 ppm, from about 0.01 ppm to about15 ppm, from about 0.01 ppm to about 10 ppm, from about 0.01 ppm toabout 5 ppm, from about 0.01 ppm to about 4 ppm, from about 0.01 ppm toabout 3 ppm, from about 0.01 ppm to about 2 ppm, from about 0.01 ppm toabout 1 ppm, from about 0.01 ppm to about 1,000 ppm, from about 0.1 ppmto 1,000 ppm, from about 1 ppm to 1,000 ppm, from about 2 ppm to about1,000 ppm, from about 3 ppm to about 1,000 ppm, from about 4 ppm toabout 1,000 ppm, from about 5 ppm to about 1,000 ppm, from about 10 ppmto about 1,000 ppm, from about 15 ppm to about 1,000 ppm, from about 25ppm to about 1,000 ppm, from about 50 ppm to about 1,000 ppm, from about75 ppm to about 1,000 ppm, from about 100 ppm to about 1,000 ppm, fromabout 150 ppm to about 1,000 ppm, from about 250 ppm to about 1,000 ppm,from about 250 ppm to about 1,000 ppm, from about 500 ppm to about 1,000ppm or from about 750 ppm to about 1,000 ppm, and values in between.

In certain embodiments of the present application, the flavorcomposition is admixed with a pet food product wherein the flavorcomposition is present in an amount of from about 0.01 ppm to about 500ppm, or from about 0.1 ppm to about 500 ppm, or from about 1 ppm toabout 500 ppm, and values in between.

In certain embodiments of the present application, the flavorcomposition is admixed with a pet food product wherein the flavorcomposition is present in an amount of from about 0.01 ppm to about 100ppm, or from about 0.1 ppm to about 100 ppm, or from about 1 ppm toabout 100 ppm, and values in between.

In certain embodiments, the flavor composition is present in the petfood product at an amount greater than about 0.01 ppm, greater thanabout 0.1 ppm, greater than about 1 ppm, greater than about 2 ppm,greater than about 3 ppm, greater than about 4 ppm, greater than about 5ppm, greater than about 10 ppm, greater than about 25 ppm, greater thanabout 50 ppm, greater than about 75 ppm, greater than about 100 ppm,greater than about 250 ppm, greater than about 500 ppm, greater thanabout 750 ppm or greater than about 1000 ppm, and values in between.

In certain embodiments, a compound of the present disclosure is presentin a food product in an amount that is sufficient to modulate, activateand/or enhance a fatty acid receptor, e.g., a GPR120 receptor. Forexample, but not by way of limitation, a compound can be present in afood product in an amount from about 10 pM to about 1 M, from about 1 nMto about 1 M, from about 1 μM to about 1 M, from about 1 mM to about 1M, from about 10 mM to about 1 M, from about 100 mM to about 1 M, fromabout 250 mM to about 1 M, from about 500 mM to about 1 M, from about750 mM to about 1 M, from about 0.001 μM to about 1 M, from about 0.001μM to about 750 mM, from about 0.001 μM to about 500 mM, from about0.001 μM to about 250 mM, from about 0.001 μM to about 100 mM, fromabout 0.001 μM to about 50 mM, from about 0.001 μM to about 25 mM, fromabout 0.001 μM to about 10 mM, from about 0.001 μM to about 1 mM, fromabout 0.001 μM to about 100 μM or from about 0.001 μM to about 10 μM,and values in between.

In certain embodiments of the present application, the flavorcomposition is admixed with a pet food product wherein the flavorcomposition is present in an amount of from about 10 pM to about 1 M, orfrom about 1 pM to about 1 M, or from about 0.1 pM to about 1 M, andvalues in between.

In certain embodiments of the present application, the flavorcomposition is admixed with a pet food product wherein the flavorcomposition is present in an amount of from about 10 pM to about 0.5 M,or from about 1 pM to about 0.5 M, or from about 0.1 pM to about 0.5 M,and values in between.

In certain embodiments of the present application, the flavorcomposition is admixed with a pet food product wherein the flavorcomposition is present in an amount of from about 10 pM to about 0.1 M,or from about 1 pM to about 0.1 M, or from about 0.1 pM to about 0.1 M,and values in between.

In certain embodiments of the present application, the flavorcomposition is admixed with a food product wherein the flavorcomposition is present in an amount of from about 0.001 to about 10%weight/weight (w/w) of the food product. For example, but not by way oflimitation, the flavor composition can be present in the amount fromabout 0.001% to about 10%, from about 0.01% to about 20% w/w, from about0.001% to about 1%, from about 0.001% to about 0.1% , from about 0.001%to about 0.01%, from about 0.01% to about 10%, from about 0.01% to about1%, or from about 0.1% to about 10%, or from about 0.1% to about 1% w/w,and values in between.

In certain embodiments of the present application, the flavorcomposition is admixed with a food product wherein the flavorcomposition is present in an amount of from about 0.001% to about 5%, orfrom about 0.01% to about 5%, or from about 0.1% to about 5% w/w, andvalues in between.

In certain embodiments of the present application, the flavorcomposition is admixed with a food product wherein the flavorcomposition is present in an amount of from about 0.001% to about 1%, orfrom about 0.01% to about 1%, or from about 0.1% to about 1% w/w, andvalues in between.

In certain embodiments of the present application, the flavorcomposition is admixed with a food product wherein the flavorcomposition is present in an amount of from about 0.01% to about 10%w/w.

In certain embodiments, the compounds of the present application areblended together in various ratios or are blended together with othercompounds, e.g., nucleotides and/or nucleotide derivatives and/orfuranones and/or amino acids and/or transmembrane compounds, to formvarious flavor compositions. Non-limiting examples of nucleotides,nucleotide derivatives, furanones, amino acids and transmembranecompounds are disclosed in International Application Nos.PCT/EP2013/072788 filed Oct. 31, 2013, PCT/EP2013/072789 filed Oct. 31,2013, PCT/EP2013/072790 filed Oct. 31, 2013, PCT/EP2013/072794 filedOct. 31, 2013, PCT/US15/65046 filed Dec. 10, 2015, PCT/US15/65036 filedDec. 10, 2015, and PCT/US15/65067 filed Dec. 10, 2015, which areincorporated herein by reference in their entireties.

5.1 Nucleotides and Nucleotide Derivatives

In certain embodiments of the present disclosure, the flavor compositioncomprises at least one compound and at least one nucleotide and/ornucleotide derivative as described herein and by InternationalApplication No. PCT/US15/65046 filed Dec. 10, 2015, which isincorporated herein by reference in its entirety.

In certain embodiments of the present disclosure, the flavor compositioncomprises at least one compound and at least two, three, four, five ormore nucleotide and/or nucleotide derivatives as described herein.Non-limiting examples of nucleotides include guanosine monophosphate(GMP), inosine monophosphate (IMP), adenosine monophosphate (AMP),cytidine monophosphate (CMP), thymine monophosphate (TMP), xanthosinemonophosphate (XMP), uridine monophosphate (UMP) and combinationsthereof.

In certain embodiments, the flavor composition can include a nucleotideand/or nucleotide derivative present in a food product which can bepresent in an amount of from about 1 pM to about 1 M, from about 1 nM toabout 1 M, from about 1 μM to about 1 M, from about 1 mM to about 1 M,from about 10 mM to about 1 M, from about 100 mM to about 1 M, fromabout 250 mM to about 1 M, from about 500 mM to about 1 M, from about750 mM to about 1 M, from about 1 μM to about 1 M, from about 1 μM toabout 750 mM, from about 1 μM to about 500 mM, from about 1 μM to about250 mM, from about 1 μM to about 100 mM, from about 1 μM to about 50 mM,from about 1 μM to about 25 mM, from about 1 μM to about 10 mM, fromabout 1 μM to about 1 mM, from about 1 μM to about 100 μM or from about1 μM to about 10 μM, and values in between.

In certain embodiments, the nucleotide and/or nucleotide derivative canbe present in an amount of greater than about 1 mM or greater than about2.5 mM of the pet food product. In certain non-limiting embodiments, thenucleotide and/or nucleotide derivative can be present in an amount ofless than about 100 mM, less than about 50 mM, less than about 20 mM orless than about 10 mM of the pet food product. In a certain,non-limiting embodiments, the nucleotide and/or nucleotide derivative ispresent in an amount of about 5 mM of the pet food product.

In certain embodiments of the present disclosure, the flavor compositionfurther comprises at least one amino acid as described herein.

In certain embodiments of the present disclosure, the flavor compositionfurther comprises at least one transmembrane compound as describedherein.

5.2 Amino Acids

In certain embodiments of the present disclosure, the flavor compositioncomprises at least one compound and at least one amino acid as describedherein, and by International Application Nos. PCT/EP2013/072788 filedOct. 31, 2013, PCT/EP2013/072789 filed Oct. 31, 2013, PCT/EP2013/072790filed Oct. 31, 2013, and PCT/EP2013/072794 filed Oct. 31, 2013, each ofwhich is incorporated herein by reference in its entirety. In certainembodiments, the flavor composition comprises at least one compound andat least two, three, four, five or more amino acids as described herein.

In certain embodiments, the flavor composition comprises at least onecompound, at least one, two, three, four, five or more first amino acidsand/or at least one, two, three, four, five or more second amino acids.

In certain embodiments of the present disclosure, the flavor compositioncomprises at least one compound, at least one first amino acid and atleast one second amino acid.

In certain embodiments of the present disclosure, the flavor compositioncomprises at least one compound, at least two first amino acids and atleast one second amino acid.

In certain embodiments of the present disclosure, the flavor compositioncomprises at least one compound, at least one first amino acid and atleast two second amino acids.

In certain embodiments of the present disclosure, the flavor compositioncomprises at least one compound, at least two first amino acids and atleast two second amino acids.

In certain embodiments of the present disclosure, the flavor compositionfurther comprises at least one fatty acid as described herein.

In certain embodiments of the present disclosure, the flavor compositionfurther comprises at least one transmembrane compound as describedherein.

Non-limiting examples of a first amino acid include tryptophan,phenylalanine, histidine, glycine, cysteine, alanine, tyrosine, serine,methionine, asparagine, leucine and combinations thereof.

Non-limiting examples of a second amino acid include threonine,isoleucine, proline, glutamic acid, aspartic acid, hydroxyl proline,arginine, cystine, glutamine, lysine, valine, orthinine, taurine,monosodium glutamate (MSG) and combinations thereof.

In certain embodiments, the at least one first amino acid and/or thesecond amino acid, alone or in combination, can be present in an amountof from about 1 mM to about 1 M, or from about 250 mM to about 1 M, orfrom about 5 mM to about 500 mM, or from about 10 mM to about 100 mM, orfrom about 15 mM to about 50 mM, or from about 20 mM to about 40 mM ofthe pet food product. In certain embodiments, the amino acid(s) can bepresent at an amount less than about 1 M, less than about 200 mM, lessthan about 100 mM, less than about 50 mM, less than about 20 mM or lessthan about 10 mM of the pet food product. In certain embodiments, thefirst amino acid and/or the second amino acid, alone or in combination,can be present in an amount of about 25 mM of the pet food product.

5.3 Transmembrane Compounds

In certain embodiments of the present disclosure, the flavor compositioncomprises at least one compound as described by the present application,and at least one transmembrane compound as described by InternationalApplication No. PCT/US15/65036 filed Dec. 10, 2015, which isincorporated herein by reference in its entirety, and in Table 1 below.

In certain embodiments of the present disclosure, the flavor compositioncomprises at least one compound and at least two, three, four, five ormore transmembrane compound.

In certain embodiments of the present disclosure, the flavor compositionincludes at least one compound, at least one transmembrane compound andat least one amino acid as described herein.

In certain embodiments of the present disclosure, the flavor compositioncomprises at least one compound, at least one transmembrane compound, atleast one first amino acid and at least one second amino acid asdescribed herein.

In certain embodiments of the present disclosure, the flavor compositionincludes at least one compound, at least one transmembrane compound andat least one nucleotide and/or nucleotide derivative as describedherein.

In certain embodiments of the present disclosure, the flavor compositionincludes at least one compound, at least one transmembrane compound, atleast one nucleotide and/or nucleotide derivative and at least one aminoacid.

In certain embodiments of the present disclosure, the flavor compositionincludes at least one compound, at least one transmembrane compound, atleast one nucleotide and/or nucleotide derivative, at least one firstamino acid and at least one second amino acid.

In certain embodiments, a transmembrane compound of the presentdisclosure can be present in a food product in an amount from about 1 pMto about 1 M, from about 1 nM to about 1 M, from about 1 μM to about 1M, from about 1 mM to about 1 M, from about 10 mM to about 1 M, fromabout 100 mM to about 1 M, from about 250 mM to about 1 M, from about500 mM to about 1 M, from about 750 mM to about 1 M, from about 1 μM toabout 1 M, from about 1 μM to about 750 mM, from about 1 μM to about 500mM, from about 1 μM to about 250 mM, from about 1 μM to about 100 mM,from about 1 μM to about 50 mM, from about 1 μM to about 25 mM, fromabout 1 μM to about 10 mM, from about 1 μM to about 1 mM, from about 1μM to about 100 μM or from about 1 μM to about 10 μM, and values inbetween.

In certain embodiments, the transmembrane compound can be a salt,stereoisomer or a comestible fours of a transmembrane compound describedherein.

TABLE 1 Compound Name 5-bromo-N-(pentan-3-yl)furan-2-carboxamide3,4-dimethyl-N-(pentan-3-yl)benzamide; N-(1-ethylpropyl)-3,4-dimethylbenzamide 4-methoxy-3-methyl-N-(1-phenylethyl)benzamideN-(2,3-dimethylcyclohexyl) benzo[d][1,3]dioxole-5-carboxamide ethyl2-(benzo[d][1,3] dioxole-5-carboxamido)-3-methylbutanoate; ethylN-(1,3-benzodioxol-5 ylcarbonyl) valinate4-methoxy-3-methyl-N-(pentan-2-yl)benzamideN-(pentan-3-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide3,4-dimethoxy-N-(pentan-3-yl)benzamide (E)-methyl3-methyl-2-(3-(thiophen-2-yl) acrylamido)butanoate(E)-3-(3,4-dimethoxyphenyl)-N-(pentan-2-yl)acrylamide; (2E)-3-(3,4-dimethoxyphenyl)-N-(pentan-2-yl)prop-2-enamide(E)-3-(4-methoxyphenyl)-N-(2-methylcyclohexyl) acrylamideN1-phenethyl-N2-(pyridin-3-ylmethyl)oxalamide; N-(2-phenylethyl)-N′-(pyridin-3-ylmethyl)ethanediamideN-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide1-(2-bromophenyl)-3-((1R,2S)-2-hydroxy-2,3-dihydro-1H-inden-1-yl)ureaN-(benzo[d][1,3]dioxol-5-yl)-2-propylpentanamide1-benzyl-3-(2-oxo-2-phenylethyl)imidazolidine-2,4,5-trioneN-(2-amino-2-oxo-1-phenylethyl)-3-chloro-4,5-dimethoxybenzamide(E)-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide2-((5-(4-(methylthio)phenyl)-2H-tetrazol-2-yl)methyl)pyridineN-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide

6. Delivery Systems

In certain embodiments, the flavor compositions of the presentapplication can be incorporated into a delivery system for use in petfood products. Delivery systems can be a non-aqueous liquid, solid, oremulsion. Delivery systems are generally adapted to suit the needs ofthe flavor composition and/or the pet food product into which the flavorcomposition will be incorporated.

The flavoring compositions can be employed in non-aqueous liquid form,dried form, solid form and/or as an emulsion. When used in dried form,suitable drying means such as spray drying can be used. Alternatively, aflavoring composition can be encapsulated or absorbed onto waterinsoluble materials. The actual techniques for preparing such driedforms are well-known in the art, and can be applied to the presentlydisclosed subject matter.

The flavor compositions of the presently disclosed subject matter can beused in many distinct physical forms well known in the art to provide aninitial burst of taste, flavor and/or texture; and/or a prolongedsensation of taste, flavor and/or texture. Without being limitedthereto, such physical forms include free forms, such as spray dried,powdered, and beaded forms, and encapsulated forms, and mixturesthereof.

In certain embodiments, the compounds of a flavor composition can begenerated during the processing of a pet food product, e.g.,sterilization, retorting and/or extrusion, from precursor compoundspresent in the pet food product. In a non-limiting example, thecomposition can be a free fatty acid that is liberated from one or moretriglycerides during the processing of a pet food product. In certainembodiments, the free fatty acid is produced during thermal, acid and/orenzymatic treatment of the triglycerides. In certain embodiments, thetriglycerides are present in meat (e.g., chicken, beef, pork, fish,and/or tallow) and/or vegetable (e.g., oils such as vegetable andsunflower oil) sources that are processed to produce the pet food.

In certain embodiments, as noted above, encapsulation techniques can beused to modify the flavor systems. In certain embodiments, flavorcompounds, flavor components or the entire flavor composition can befully or partially encapsulated. Encapsulating materials and/ortechniques can be selected to determine the type of modification of theflavor system.

In certain embodiments, the encapsulating materials and/or techniquesare selected to improve the stability of the flavor compounds, flavorcomponents or flavor compositions; while in other embodiments theencapsulating materials and/or techniques are selected to modify therelease profile of the flavor compositions.

Suitable encapsulating materials can include, but are not limited to,hydrocolloids such as alginates, pectins, agars, guar gums, celluloses,and the like, proteins, polyvinyl acetate, polyethylene, crosslinkedpolyvinyl pyrrolidone, polymethylmethacrylate, polylactidacid,polyhydroxyalkanoates, ethylcellulose, polyvinyl acetatephthalate,polyethylene glycol esters, methacrylicacid-co-methylmethacrylate,ethylene-vinylacetate (EVA) copolymer, and the like, and combinationsthereof. Suitable encapsulating techniques can include, but are notlimited to, spray coating, spray drying, spray chilling, absorption,adsorption, inclusion complexing (e.g., creating a flavor/cyclodextrincomplex), coacervation, fluidized bed coating or other process can beused to encapsulate an ingredient with an encapsulating material.

Encapsulated delivery systems for flavoring agents or sweetening agentscan contain a hydrophobic matrix of fat or wax surrounding a sweeteningagent or flavoring agent core. The fats can be selected from any numberof conventional materials such as fatty acids, glycerides or polyglycerol esters, sorbitol esters, and mixtures thereof. Examples offatty acids include but are not limited to hydrogenated and partiallyhydrogenated vegetable oils such as palm oil, palm kernel oil, peanutoil, rapeseed oil, rice bran oil, soybean oil, cottonseed oil, sunfloweroil, safflower oil and combinations thereof. Examples of glyceridesinclude, but are not limited to, monoglycerides, diglycerides andtriglycerides.

Waxes can be chosen from the group consisting of natural and syntheticwaxes and mixtures thereof. Non-limiting examples include paraffin wax,petrolatum, carbowax, microcrystalline wax, beeswax, carnauba wax,candellila wax, lanolin, bayberry wax, sugarcane wax, spermaceti wax,rice bran wax, and mixtures thereof.

The fats and waxes can be use individually or in combination in amountsvarying from about 10 to about 70%, and alternatively in amounts fromabout 30 to about 60%, by weight of the encapsulated system. When usedin combination, the fat and wax can be present in a ratio from about70:10 to 85:15, respectively.

Typical encapsulated flavor compositions, flavoring agent or sweeteningagent delivery systems are disclosed in U.S. Pat. Nos. 4,597,970 and4,722,845, the disclosures of which are incorporated herein by referencein their entireties.

Liquid delivery systems can include, but are not limited to, systemswith a dispersion of the flavor compositions of the present application,such as in carbohydrate syrups and/or emulsions. Liquid delivery systemscan also include extracts where the compound and/or the flavorcompositions are solubilized in a solvent. Solid delivery systems can becreated by spray drying, spray coating, spray chilling, fluidized beddrying, absorption, adsorption, coacervation, complexation, or any otherstandard technique. In some embodiments, the delivery system can beselected to be compatible with or to function in the edible composition.In certain embodiments, the delivery system will include an oleaginousmaterial such as a fat or oil. In certain embodiments, the deliverysystem will include a confectionery fat such as cocoa butter, a cocoabutter replacer, a cocoa butter substitute, or a cocoa butterequivalent.

When used in dried form, suitable drying means such as spray drying maybe used. Alternatively, a flavoring composition may be adsorbed orabsorbed onto substrates, such as water insoluble materials, and may beencapsulated. The actual techniques for preparing such dried forms arewell known in the art.

7. Pet Food Products

The flavor compositions of the present disclosed subject matter can beused in a wide variety of pet food products. Non-limiting examples ofsuitable pet food products include wet food products, dry food products,moist food products, pet food supplements (e.g., vitamins), pet beverageproducts, snack and treats as described herein.

The combination of the flavoring composition(s) of the presentlydisclosed subject matter together with a pet food product and optionalingredients, when desired, provides a flavoring agent that possessesunexpected taste and imparts, for example, a fatty acid sensoryexperience, for example, through an increase in activity of a GPR120fatty acid receptor. The flavor compositions disclosed herein can beadded prior to, during or after formulation processing or packaging ofthe pet food product, and the components of the flavor composition canbe added sequentially or simultaneously. In certain embodiments, thecompounds of a flavor composition can be generated during the processingof a pet food product, e.g., sterilization, retorting and/or extrusion,from precursor compounds present in the pet food product. In anon-limiting example, the composition can be a free fatty acid that isliberated from one or more triglycerides during the processing of a petfood product. In certain embodiments, the free fatty acid is producedduring thermal, acid and/or enzymatic treatment of the triglycerides. Incertain embodiments, the triglycerides are present in meat (e.g.,chicken, beef, pork, fish, and/or tallow) and/or vegetable (e.g., oilssuch as vegetable and sunflower oil) sources that are processed toproduce the pet food.

In certain embodiments, the pet food product is a nutritionally completedry food product. A dry or low moisture-containingnutritionally-complete pet food product can comprise less than about 15%moisture, and include from about 10 to about 60% fat, from about 10% toabout 70% protein and from about 30% to about 80% carbohydrates, e.g.,dietary fiber and ash.

In certain embodiments, the pet food product is a nutritionally completewet food product. A wet or high moisture-containingnutritionally-complete pet food product can comprise greater than about50% moisture. In certain embodiments, the wet pet food product includesfrom about 40% fat, from about 50% protein and from about 10%carbohydrates, e.g., dietary fiber and ash.

In certain embodiments, the pet food product is a nutritionally completemoist food product. A moist, e.g., semi-moist or semi-dry or soft dry orsoft moist or intermediate or medium moisture containingnutritionally-complete pet food product comprises from about 15 to about50% moisture.

In certain embodiments, the pet food product is a pet food snackproduct. Non-limiting examples of pet food snack products include snackbars, pet chews, crunchy treats, cereal bars, snacks, biscuits and sweetproducts.

In certain embodiments, the protein source can be derived from a plantsource, such as lupin protein, wheat protein, soy protein andcombinations thereof. Alternatively or additionally, the protein sourcecan be derived from a variety of animal sources. Non-limiting examplesof animal protein include beef, pork, poultry, lamb, or fish including,for example, muscle meat, meat byproduct, meat meal or fish meal.

8. Methods of Measuring Taste Attributes

In certain embodiments of the present disclosure, the taste, flavorand/or palatability attributes of a pet food product can be modified byadmixing a flavor composition with the food product, or generated underfood preparation conditions, as described herein. In certainembodiments, the attribute(s) can be enhanced or reduced by increasingor decreasing the concentration of the flavor composition admixed orgenerated with the food product. In certain embodiments, the tasteattributes of the modified food product can be evaluated as describedherein, and the concentration of flavor composition admixed or generatedwith the food product can be increased or decreased based on the resultsof the evaluation.

In certain embodiments of the present disclosure, the taste and/orpalatability attributes can be measured using an in vitro assay, whereina compound's ability to activate a feline GPR120 receptor expressed bycells in vitro at different concentrations is measured. In certainembodiments, an increase in the activation of the receptor correlateswith an increase in the taste and/or palatability attributes of thecompound. In certain embodiments, the composition is measured alone orin combination with other compounds. In certain embodiments the in vitroassay comprises the in vitro assays described in the Examples section ofthe present application.

In certain embodiments of the present disclosure, the taste and/orpalatability attributes can be measured using a panelist of tastetesters. For example, but not by way of limitation, the panel cancontain feline panelists. In certain embodiments, the panel can includecanine panelists. In certain embodiments, the palatability of a pet foodproduct can be determined by the consumption of a pet food productcontaining a flavor composition alone (e.g., the one bowl test, monadicranking). In certain embodiments, the palatability of a pet food productcan be determined by the preferential consumption of a pet food productcontaining a flavor composition, disclosed herein, versus a pet foodproduct that does not contain the flavor composition or another flavorcomposition (e.g., the two bowl test for testing preference, differenceand/or choice).

In certain embodiments, the palatability and/or fatty acid taste of aflavor composition can be determined by the preferential consumption ofan emulsion (or any other composition into which a compound of thepresent application can be admixed with, for example, a gel or gelatin)containing a flavor composition, disclosed herein, versus an emulsionthat does not contain the flavor composition or contains a differentflavor composition. For example, a gel panel can be used to compare thepalatability of a range of concentrations of compounds in a monadicexposure. In certain embodiments, the emulsion can contain apalatability enhancer, for example, L-histidine, as aningestive/positive tastant to increase baseline emulsion intake,therefore enabling the identification of a potential negative impact ofthe test compound.

The intake ratio for each pet food product or emulsion can be determinedby measuring the amount of one ration consumed divided by the totalconsumption. The consumption ratio (CR) can then be calculated tocompare the consumption of one ration in terms of the other ration todetermine the preferential consumption of one food product or emulsionover the other. Alternatively or additionally, the difference in intake(g) can be used to assess the average difference in intake between thetwo emulsions in a two bottle test or between two pet food products in atwo bowl test at a selected significance level, for example, at the 5%significance level to determine an average difference in intake with a95% confidence interval. However, any significance level may be used,for example, a 1, 2, 3, 4, 5, 10, 15, 20, 25, or 50% significance level.In certain embodiments, percentage preference scores, e.g., thepercentage preference for one emulsion or food product by an animal isthe percentage of the total emulsion or food product ingested during thetest that that emulsion or food product accounts for, can also becalculated.

9. Methods of Generation

In certain embodiments, the compounds of the present disclosure can begenerated using standard chemosynthesis processes. In certainembodiments, the chemosynthesis process provides a compound having apurity of at least 99.999%, or at least 99%, or at least 95%, or atleast 90%, or at least 85 or at least 80%. In certain embodiments, thecompounds can be prepared using standard hydrolysis processes such asthose employing acids, enzymes or a combination of acids and enzymes.

In certain embodiments, the compounds of the present disclosure can begenerated under food preparation conditions, e.g., during the productionof a pet food product. For example, but not by way of limitation, thecompounds of the present disclosure can be generated during a thermalfood process, e.g., sterilization, retorting and/or extrusion, fromprecursor compounds present in the pet food. In certain embodiments, aliquid and/or a powder palatant can also be added to enhance the tasteof a pet food, e.g., to a dry pet food product, and to increase thepalatability of the pet food. The palatant can be a digest of meat(e.g., liver) and/or a digest of a vegetable, and can optionally includeother palatants known in the art. In certain embodiments, the compoundcan be admixed with or generated in the liquid and/or powder palatantprior to its addition to the pet food product. Alternatively oradditionally, the compound can be admixed with or generated in theliquid and/or powder palatant after its addition to the pet foodproduct.

10. Non-limiting Examples of Methods of the Present Disclosure

In certain non-limiting embodiments, the present disclosure provides fora method of increasing the palatability of a pet food product comprisingadmixing the pet food product with a flavor composition comprising acompound as described herein, wherein the compound is present at aconcentration of from about 10 pM to about 1 M in the admixture.

In certain non-limiting embodiments, the present disclosure provides fora method of increasing the palatability of a pet food product comprisingproducing the pet food product with a flavor composition comprising acompound as described herein, wherein the compound is present at aconcentration of from about 10 pM to about 1 M in the product.

In certain non-limiting embodiments, the present disclosure provides fora method of increasing the fatty acid taste of a pet food product, forexample, by increasing the activity of a GPR120 receptor, comprisingadmixing the pet food product with a flavor composition comprising acompound as described herein, wherein the compound is present at aconcentration of from 0.01 ppm to 1,000 ppm in the admixture.

In certain non-limiting embodiments, the present disclosure provides fora method of increasing the palatability of a pet food product comprisingadmixing the pet food product with a flavor composition comprising acompound as described herein, wherein the flavor composition is presentat a concentration of from about 0.01 ppm to 1,000 ppm in the admixture.

In certain non-limiting embodiments, the present disclosure provides fora method of increasing the fatty acid taste of a pet food product, forexample, by increasing the activity of a GPR120 receptor, comprisingadmixing the pet food product with a flavor composition comprising acompound as described herein, wherein the flavor composition is presentat a concentration of from about 0.001% to about 10% w/w, or from about0.01% to about 20% w/w, or from about 0.01% to about 5% w/w, or fromabout 0.01% to about 1% w/w in the admixture.

In certain non-limiting embodiments, the present disclosure provides fora method of increasing the palatability of a pet food product comprisingadmixing the pet food product with a flavor composition comprising acompound as described herein, wherein the flavor composition is presentat a concentration of from about 0.001% to about 10% w/w, or from about0.01% to about 20% w/w, or from about 0.01% to about 5% w/w, or fromabout 0.01% to about 1% w/w in the admixture.

EXAMPLES

The presently disclosed subject matter will be better understood byreference to the following Examples, which are provided as exemplary ofthe invention, and not by way of limitation.

Example 1 Compounds that Activate the fGPR120 Receptor

The present example describes the activation of the cat GPR120 receptor(fGPR120) by compounds in vitro.

Compounds that may function as GPR120 agonists were identified by invitro functional characterization. The effectiveness of a compound inactivating the fGPR120 receptor was evaluated.

Methods: CHO-K1 cells that stably express fGPR120 were used to screenabout 120 test compounds to identify compounds that activate the fGPR120fatty acid receptor. Activation of the fGPR120 receptor was detected bya change in intracellular calcium levels using a calcium sensitivefluorescent dye. Cells that do not express the fGPR120 receptor (mockcells) were used as a control. A FLIPR® Tetra was used for data capture.

Each compound was first tested for its ability to activate fGPR120 atthree different dose levels (10, 3 and 1 μM; 50, 16.7, and 5.6 μM; or100, 30 and 10 μM) (FIGS. 1A-1AH). Dose response curves were thengenerated for each compound. (FIGS. 2A-2AH). The following propertieswere determined: percent increase in fGPR120 activity induced by thecompounds compared to an fGPR120 agonist control (alpha-linolenic acid),percent increase in fGPR120 activity induced by the compounds comparedto an ATP background control, and concentration of compound required toachieve EC50 (i.e., 50% of the maximal activity achieved).

Results: Changes in fGPR120 activity following treatment of CHO-K1 cellsexpressing fGPR120 receptors, or CHO-K1 cells not expressing fGPR120(Mock control cells), with 34 active compounds that increased theactivity of fGPR120 by at least 30% compared to control are shown inFIGS. 1A-1AH and 2A-2AH. Table 2 identifies the 34 compounds thatincreased the activity of fGPR120 by at least 30% compared to control.

TABLE 2 GPR120 binding compounds that increased the activity of GPR120by at least 30% compared to control. No. Compound 1.(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoic acid (also known asArachidonic Acid) 2. (5Z,8Z)-icosa-5,8-dienoic acid 3.4-[4-(heptyloxy)phenyl]-4-oxobutanoic acid 4. (11Z)-octadec-11-enoicacid (also known as cis-Vaccenic acid) 5. (9E)-hexadec-9-enoic acid(also known as Palmitelaidic acid) 6. tridec-12-enoic acid (also knownas 12-Tridecenoic acid) 7. S-Farnesyl Thioacetic Acid 8.(10Z)-pentadec-10-enoic acid (also known as (10Z)-10-Pentadecenoic acid)9. 10(E),12(Z)-Conjugated Linoleic Acid (also known as (10Z,12Z)-10,12-Octadecadienoic acid) 10. (10Z,13Z)-nonadeca-10,13-dienoic acid 11.(9Z,11E)-octadeca-9,11-dienoic acid 12. cis-7-Hexadecenoic Acid 13.dodecanoic acid (also known as Lauric acid) 14. (9Z)-tetradec-9-enoicacid (also known as Myristoleic acid) 15.(11Z,14Z,17Z)-icosa-11,14,17-trienoic acid (also known asDihomo-α-linolenic acid (20:3(n-3))) 16.(6Z,9Z,12Z)-octadeca-6,9,12-trienoic acid (also known as γ-Linolenicacid) 17. (11Z,14Z)-icosa-11,14-dienoic acid (also known asDihomolinoleic acid (20:2(n-6))) 18. (9Z)-hexadec-9-enoic acid (alsoknown as palmitoleate, (Z)-Palmitoleic acid) 19. 12-methoxydodecanoicacid 20. (8Z,11Z,14Z)-icosa-8,11,14-trienoic acid 21.(9Z,12Z)-octadeca-9,12-dienoic acid (also known as Linoleic acid) 22.(10Z)-heptadec-10-enoic acid 23. Pinolenic Acid 24.(9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid (also known as α-Linolenicacid) 25. tridecanoic acid (also known as Tridecylic acid) 26.tetradecanoic acid (also known as Myristic acid) 27.(9Z)-octadec-9-enoic acid (also known as Oleic acid) 28. GW 9508 (alsoknown as 4-[[(3-Phenoxyphenyl)methyl]amino]benzenepropanoic acid) 29.(5Z,8Z,11Z,14Z,17Z)-icosa-5,8,11,14,17-pentaenoic acid (also known asEicosapentanoic acid) 30.3-(4-((4-fluoro-4′-methylbiphenyl-2-yl)methoxy)phenyl)propanoic acid(also known as TUG 891 and4-[(4-Fluoro-4′-methyl[1,1′-biphenyl]-2-yl)methoxy]- benzenepropanoicacid) 31. (10E)-pentadec-10-enoic acid 32. (9E)-tetradec-9-enoic acid(also known as Myristoleate) 33.(6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraenoic acid 34.(5Z,8Z,11Z)-icosa-5,8,11-trienoic acid (also known as Mead acid)

Example 2 Identification of GPR120 Agonists using In Silico Screening

Site-directed mutagenesis has been used to identify amino acid residuesof human GPR120 that interact with GPR120 binding compounds (Hudson etal.). However, the present example describes the computational modelingof the feline and canine GPR120 that was conducted to identify putativecompound agonists of the GPR120 receptor. GPR120 belongs to theG-Protein Coupled Receptor (GPCR) group A family of receptors. Thefeline GPR120 exhibits 96% sequence identity to the canine GPR120, and89% sequence identity to human GPR120. (See, for example, InternationalPublication No. WO 2014/199114, published Dec. 18, 2014, which isincorporated by reference in its entirety).

Computational approaches were used to analyze the three-dimensionalstructure of the GPR120 receptor to identify polypeptide regions thatcan be exploited to selectively activate the receptor. A structuralhomology model of the GPR120 receptor was generated based on thestructures of class A GPCRs. The homology models were built inSWISS-MODEL and GPCR-I-TASSER web based environments and further refinedin the Discovery Studio (DS) suite of programs from Accelrys (BIOVIA,Dassault Systèmes). Modeler program from DS was used for modelrefinement (see Eswar et al., Current Protocols in Bioinformatics,Supplement 15:5.6.1-5.6.30 (2006), which is incorporated by referenceherein in its entirety). “In silico” screening was used to identifycompounds that interact with the GPR120.

The GPCR group A family of proteins includes numerous solved crystalstructures. Homology models of feline and canine GPR120 transmembranedomain was generated based on the crystal structures of GPCR Group Afrom Protein Data Bank (PDB): 4DJH (human kappa opioid receptor (Wu, H.et al.)), 3OE6 (CXCR4 chemokine receptor (Wu, B. et al.)) and 3SN6(beta2 adrenergic receptor (Rasmussen et al.)). The docking program,BioDock, from BioPredict was used to dock the compounds3-(4-((4-fluoro-4′-methylbiphenyl-2-yl)methoxy)phenyl)propanoic acid(also known as TUG891) (FIGS. 4A-4C);4-[4-(heptyloxy)phenyl]-4-oxobutanoic acid (FIGS. 5A-5C);eicosapentanoic acid (FIGS. 6A-6C); oleic acid (FIGS. 8A and 8B); andlinoleic acid (FIGS. 9A-9C) into the active site of the transmembranedomain of the feline GPR120, in silico. These compounds were also dockedinto the active site of the transmembrane domain of the canine GPR120,in silico. Active sites of the human, feline and canine GPCR are similarin structure.

Based on the in silico binding models, the compounds were observed tohave the following potential interactions with the amino acids of thetransmembrane domain of the feline GPR120:

PHE88, VAL95, VAL98 and ARG99 on Helix 2;

PHE115, MET118, SER119, GLY122, SER123 on Helix 3;

TRP207, PHE211, VAL212, ASN215 on Helix 5;

TRP277, ILE280, ILE281, ILE284 on Helix 6;

TRP299, PHE303, PHE304, VAL307, THR310, PHE311 on Helix 7.

Many of the interactions between the compounds and the feline GPR120active site involve GPR120 hydrophobic residues. Additionally, there isa salt bridge and hydrogen bond between the carboxyl groups of thecompounds and ARG99.

REFERENCES

-   -   1. Rasmussen, S. G. et al., “Crystal structure of the beta2        adrenergic receptor-Gs protein complex.” (2011) Nature 477:        549-555.    -   2. Wu, B. et al., “Structures of the CXCR4 chemokine GPCR with        small-molecule and cyclic peptide antagonists.” (2010) Science        330: 1066-1071.    -   3. Wu, H. et al., “Structure of the human kappa-opioid receptor        in complex with JDTic.” (2012) Nature 485: 327-332.    -   4. Brian D. Hudson et al., “The Molecular Basis of Ligand        Interaction at Free Fatty Acid Receptor 4 (FFA4/GPR120).” The        Journal of Biological Chemistry, 2014 Jul. 18; 289(29):20345-58.    -   5. Arnold K, Bordoli L, Kopp J, and Schwede T., “The SWISS-MODEL        Workspace: A web-based environment for protein structure        homology modeling.” (2006) Bioinformatics., 22,195-201.    -   6. Zhang, J Yang, R Jang, Y Zhang., “GPCR-I-TASSER: A hybrid        approach to G protein-coupled receptor structure modeling and        the application to the human genome.” Structure, 23: 1538-1549,        2015 20345-20358, Jul. 18, 2014.

Example 3 Identification of Dodecyl Dihydrogen Phosphate as a PositiveAllosteric Modulator of GPR120 using In Vitro Screening

The present example describes an in vitro assay for measuring theactivity of dodecyl dihydrogen phosphate as a GPR120 agonists andpositive allosteric modulator (PAM).

Methods: CHO cells that stably express fGPR120 were used to assaydodecyl dihydrogen phosphate for activity as an fGPR120 agonist or PAM.

CHO/NatClytin/fGpr120+Gα16 (fGpr120) cells and CHO/NatClytin/mock+Gα16(mock) cells were seeded at 5,000 cells/well. 24 hours after cellseeding, cells were loaded with 0.5× Fluo8-NW dye in standard tyrodebuffer (20 μl/well) for 1 h at room temperature. The ability of dodecyldihydrogen phosphate to activate fGPR120 expressed by the CHO cells wasdetermined by measuring fluorescence using a FLIPR® Tetra screeningsystem after contacting the cells with the compound in agonist mode andPAM mode according to the following protocol:

-   -   1. Agonist mode: injection of 3× dodecyl dihydrogen phosphate        (10 μl/well), wherein fluorescence (i.e., agonist activity) was        measured for 3 minutes.    -   2. PAM mode: injection of 3× dodecyl dihydrogen phosphate (10        μl/well), wherein fluorescence was measured for 3 min. After        about 1 additional minute, injection of 3× fGPR120 agonist at a        concentration corresponding to the agonist's EC₂₀ (15 μl/well),        wherein fluorescence (i.e., PAM activity) was measured for 3        min. The fGPR120 agonist was 1.5 μM α-linolenic acid. For mock        controls, 0.2μ ATP was used as the agonist.

The maximum of ΔF/F values measured using the FLIPR® Tetra system wereexported and used for data analysis. For PAM data analysis, data werenormalized onto EC₁₀₀ (as 100% activity) and EC₂₀ (as 0% activity)values.

The compounds used in the experiments were dissolved 6× in 0.01% BSA(fatty acid-free) and then pre-mixed on compound plates with buffer. Thefinal BSA concentration of the cells was 0.00167%.

Controls for agonist mode experiments—dose response curves ofα-linolenic acid (FIGS. 12A and 12B): 100 μM α-linolenic acid (fGpr120max signal), 30 μM ATP (max signal), buffer (min signal), buffer+0.01%BSA (min signal).

Controls for PAM mode experiments—dose response curves of α-linolenicacid (fGpr120) or ATP (mock) (FIGS. 12A and 12B): 100 μM α-linolenicacid (max signal for Gpr120), 1.5 μM α-linolenic acid (EC₂₀, min signalfor Gpr120), 30 μM ATP (max signal), 0.2 μM ATP (EC₂₀, min signal).

Results: As shown in FIGS. 11A and 11B, dodecyl dihydrogen phosphatemodulated the activity of fGPR120 as a PAM in the presence of an fGPR120agonist.

Although the presently disclosed subject matter and its advantages havebeen described in detail, it should be understood that various changes,substitutions and alterations can be made herein without departing fromthe spirit and scope of the invention as defined by the appended claims.Moreover, the scope of the present application is not intended to belimited to the particular embodiments of the process, machine,manufacture, composition of matter, means, methods and steps describedin the specification. As one of ordinary skill in the art will readilyappreciate from the disclosure of the presently disclosed subjectmatter, processes, machines, manufacture, compositions of matter, means,methods, or steps, presently existing or later to be developed thatperform substantially the same function or achieve substantially thesame result as the corresponding embodiments described herein may beutilized according to the presently disclosed subject matter.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

Patents, patent applications, publications, product descriptions andprotocols are cited throughout this application the disclosures of whichare incorporated herein by reference in their entireties for allpurposes.

We claim:
 1. A flavor composition for a cat food product comprising acompound of Formula II:

wherein X is (C)R₁R₂, O, S, C(O), or a chemical bond; X₁ is (C)R₁R₂, O,S, or N(R₁); Y is COOH, SO₃H, —OP(O)(OH)₂, or Tetrazole; W is halogen,COOH, COOMe, CN, N(R₁)(R₂), CHO, CONR₁R₂, Aryl, OH, H, —S-Aryl, —O-Aryl,—N-Aryl, CF₃, OCH₃, CH₃, NO₂, or OEt; Z₁ and Z₂ are each independentlyselected from halogen, COOH, COOMe, CN, N(R₁)(R₂), CHO, CONR₁R₂, Aryl,OH, H, —S-Aryl, —O-Aryl, —N-Aryl, CF₃, OCH₃, CH₃, NO₂, and OEt; R₁ andR₂ are each independently selected from H and branched or unbranchedC₁-C₆ lower alkyl; n₁ is 0-4; n₂ is 0-2; and n₃ is 0-4, wherein thecompound is present in the flavor composition at a concentration of fromabout 0.001 μM to about 1 mM, wherein the compound is a feline GPR 120agonist, and wherein the compound increases a fatty acid taste of thecat food product.
 2. The flavor composition of claim 1, wherein thecompound is selected from the group consisting of3-(4-((4-fluoro-4′-methylbiphenyl-2-yl)methoxy)phenyl)propanoic acid,and 4-[[(3-Phenoxyphenyl)methyl]amino]benzenepropanoic acid.
 3. A catfood product comprising the flavor composition of claim 1, wherein theflavor composition is present in an amount effective to increase thepalatability of the food product, as determined by a panel of tastetesters.
 4. The cat food product of claim 3, wherein the flavorcomposition is present at a concentration of from about 0.001% to about10% w/w in the cat food product.
 5. The cat food product of claim 3,wherein the cat food product is a wet pet food product.
 6. The cat foodproduct of claim 3, wherein the cat food product is a dry pet foodproduct.
 7. A method of increasing a fatty acid taste intensity in a catfood product comprising admixing a cat food product with a flavorcomposition comprising a compound represented by Formula II:

wherein X is (C)R₁R₂, O, S, C(O), or a chemical bond; X₁ is (C)R₁R₂, O,S, or N(R₁); Y is COOH, SO₃H, —OP(O)(OH)₂, or Tetrazole; W is halogen,COOH, COOMe, CN, N(R₁)(R₂), CHO, CONR₁R₂, Aryl, OH, H, —S-Aryl, —O-Aryl,—N-Aryl, CF₃, OCH₃, CH₃, NO₂, or OEt; Z₁ and Z₂ are each independentlyselected from halogen, COOH, COOMe, CN, N(R₁)(R₂), CHO, CONR₁R₂, Aryl,OH, H, —S-Aryl, —O-Aryl, —N-Aryl, CF₃, OCH₃, CH₃, NO₂, and OEt; R₁ andR₂ are each independently selected from H and branched or unbranchedC₁-C₆ lower alkyl; n₁ is 0-4; n₂ is 0-2; and n₃ is 0-4; wherein thecompound is present in the flavor composition at a concentration of fromabout 0.001 μM to about 1mM, wherein the compound is a feline GPR 120agonist, and wherein the compound increases a fatty acid taste of thecat food product.
 8. The method of claim 7, wherein the flavorcomposition is present at a concentration of from about 0.001% to about10% w/w in the cat food product.